Susceptibility of porcine intestine to pilus-mediated adhesion by some isolates of piliated enterotoxigenic Escherichia coli increases with age. Two porcine isolates of enterotoxigenic Escherichia coli (ETEC) (serogroup O157 and O141) derived from fatal cases of postweaning diarrhea and lacking K88, K99, F41, and 987P pili (4P- ETEC) were tested for adhesiveness to small-intestinal epithelia of pigs of different ages. Neither strain adhered to isolated intestinal brush borders of newborn (1-day-old) pigs in the presence of mannose. However, mannose-resistant adhesion occurred when brush borders from 10-day- and 3- and 6-week-old pigs were used. Electron microscopy revealed that both strains produced fine (3.5-nm) and type 1 pili at 37 degrees C but only type 1 pili at 18 degrees C. Mannose-resistant in vitro adhesion to brush borders of older pigs correlated with the presence of fine pili. These strains produced predominantly fine pili in ligated intestinal loops of both older and newborn pigs, but adherence was greater in loops in older pigs. Immunoelectron microscopic studies, using antiserum raised against piliated bacteria and absorbed with nonpiliated bacteria, of samples from brush border adherence studies revealed labelled appendages between adherent bacteria and intestinal microvilli. Orogastric inoculation of pigs weaned at 10 and 21 days of age indicated significantly (P less than 0.001) higher levels of adhesion by the ETEC to the ileal epithelia of older pigs than to that of younger ones. We suggest that small-intestinal adhesion and colonization by these ETEC isolates is dependent on receptors that develop progressively with age during the first 3 weeks after birth. Furthermore, our data are consistent with the hypothesis that the fine pili described mediate intestinal adhesion by the 4P- ETEC strains studied.


Interleukin-6 response of epithelial cell lines to bacterial stimulation in vitro. This study demonstrated that epithelial cell lines secrete interleukin-6 (IL-6) in response to stimulation with gram-negative bacteria. Human epithelial cell lines of urinary tract origin (A-498 and J82) and of intestinal origin (HT-29 and Caco-2) were analyzed for the secretion of IL-6 by using the B9 bioassay. The supernatants from cells maintained with culture medium were used to assess the constitutive production of IL-6. The supernatants from cells exposed to Escherichia coli strains, lipopolysaccharide, lipid A, and isolated fimbriae were used to quantitate the IL-6 response to these stimulants. The urinary tract epithelial cell lines were found to constitutively secrete IL-6. The IL-6 activity in the supernatants of the bladder cell line (J82) increased above constitutive levels after 2 h of stimulation by most of the bacterial strains tested. The IL-6 activity in the supernatants of the kidney line (A-498) accumulated at a constant rate over the 24-h assay period. The role of bacterial adherence for the induction of IL-6 production was investigated by comparing the responses to recombinant E. coli strains expressing different fimbriae. In addition, isolated P and S fimbriae with and without the receptor binding domain were also used as stimulants. The IL-6 activity in the supernatants of the bladder cell line increased after exposure to bacteria and bacterial products regardless of their adhesive properties. In contrast, the kidney cell line was stimulated to secrete significantly more IL-6 by adhering bacteria and by adhesin-positive P fimbriae than by nonadhering bacteria or adhesin-negative P fimbriae. The S-fimbrial preparations had no specific effects on the IL-6 activity of the cell supernatants. These results are consistent with our hypothesis that epithelial cells can be a major source of IL-6 when stimulated by bacteria and that the adhesive properties of the bacteria can influence this response.


Protection of squirrel monkeys against virulent Plasmodium falciparum infections by use of attenuated parasites. We previously showed that the Uganda Palo Alto line of Plasmodium falciparum propagated in Saimiri monkeys and the line maintained in culture in human erythrocytes for many years in our laboratory are genetically unrelated (T. Fandeur, S. Bonnefoy, and O. Mercereau-Puijalon, Mol. Biochem. Parasitol. 47:167, 1991). When injected into a splenectomized Saimiri monkey, the in vitro-derived Palo Alto population procured a long-lasting, low-grade parasitemia that was spontaneously resolved by the animal. This line was propagated by serial blood transfers in two other monkeys without enhancement of the virulence of the parasites. The genetic characteristics of parasite samples corresponding to the different passages of the line in monkeys were stable for the several markers examined (pPF11.1, MSA1, and MSA2), although microheterogeneity was detected in telomeric and subtelomeric regions of chromosomes. Interestingly, in vitro-derived Palo Alto parasites induced a strong, potent immunity that enabled the monkeys to completely block subsequent challenge with two different heterologous lethal P. falciparum lines. These attenuated parasites are thus genetically stable in monkeys and represent an attractive model for assessing the feasibility of a live attenuated malaria vaccine.


Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds. One hundred and sixty-four clinical isolates of Pasteurella multocida recovered from two swine herds in Minnesota were characterized by restriction endonuclease analysis (REA) and rRNA gene restriction fragment length patterns. Bacterial DNA was digested with HpaII and electrophoresed in 0.55% agarose. Restriction fragments were transferred by Southern blot to nylon membranes and then hybridized with digoxigenin-dUTP-labeled Escherichia coli rRNA. Four different REA patterns were observed among the 156 serotype A strains isolated from herds A and B. The two most common REA types (1 and 2) represented 92% of the strains analyzed, while REA types 3 and 4 were observed only in lung samples and accounted for 8% of the isolates. Two different ribotypes were observed for these serotype A isolates. Ribotype I consisted of the most common types, 1 and 2, found by DNA fingerprinting. Ribotype II included REA types 3 and 4. Results from both herds suggest that in closed swine populations, a single strain of P. multocida predominates and causes disease. It is concluded that these genomic fingerprinting techniques were highly discriminatory and that capsular serotyping in combination with REA or ribotyping is an appropriate technique for epidemiological studies of P. multocida of swine origin.


Xamoterol in severe congestive heart failure: long-term oral treatment, a double-blind randomised study. Twelve patients in severe congestive heart failure were given placebo, 100 mg xamoterol (Corwin) twice daily and 200 mg xamoterol twice daily, respectively, in 3 two-week periods in a double-blind randomised study. At the end of each treatment period the patients were evaluated. No differences were found between placebo and xamoterol in the following parameters: New York Heart Association function group index, heart volume, body weight, exercise duration on bicycle and treadmill, heart rate and systolic and diastolic blood pressure at rest. However, during exercise we found significantly lower heart rate and rate-pressure product during xamoterol treatment. This reduction is probably indicating occupation of beta-adrenoreceptors with concomitant reduced oxygen consumption during exercise.


Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)


[Neuron-specific enolase (NSE)--a suitable tumor marker in malignant melanoma?]. The neuron-specific enolase (NSE) level is elevated in neurons and in numerous cells of the APUD system; melanocytes are also considered to belong to this system. In order to test the relevance of NSE as a tumour marker for malignant melanoma, its concentration in serum was radioimmunologically determined in 89 patients with melanomas: 24 in stage I (primary tumours), 44 in stage II (regional metastases), and 21 in stage III (distant metastases). The average (+/- coefficient of variation) concentrations recorded were 7.4 micrograms/l (+/- 46%) in patients in stage I, 5.8 micrograms/l (+/- 32%) in those in stage II, and 11.0 micrograms/l (+/- 72%) in those in stage III. A threshold value of 11.5 micrograms/l was exceeded in 9 cases, including 8 patients in stage III. Since definitely increased values arose almost exclusively in distant metastases, determination of NSE levels in serum is hardly a suitable tool for early detection of latent metastases.


An investigation of Japanese subjects maps susceptibility to type 1 (insulin-dependent) diabetes mellitus close to the DQA1 gene. Insulin-dependent diabetic and control subjects of Japanese origin were HLA-DRB1, -DQB1, and -DQA1 typed using restriction fragment length polymorphism analysis and sequence-specific oligonucleotide gene probing. The DQA1 allele DQA1*0301 was positively associated with the disease [48/52 (92%) diabetic patients versus 44/64 (69%) control subjects, Pc less than 0.03, RR = 4.97]. Alleles of the DRB1 and DQB1 genes showed no significant association with the disease. The frequency of DQB1 genotypes encoding the amino acid aspartic acid at position 57 of the DQ beta chain did not differ significantly between subjects with insulin-dependent diabetes mellitus (IDDM) and controls. These findings suggest that a susceptibility allele for IDDM in the Japanese is more closely associated with the DQA1 gene than the DQB1 gene.


A review of ectoparasites and their effect on cattle production. Losses in livestock production due to ectoparasite infestations exceed $2.26 billion annually. Over 50 species of ectoparasites infest cattle throughout the United States. The horn fly, Haematobia irritans (L.), is the most important and widespread of the five to six major pest species of pastured cattle in the southern region. Results from the examination of production traits from cattle under ectoparasite burdens have been variable, ranging from no effect to significant reductions in weight gains. Because of this inconsistency, specific physiological and nutritional responses in cattle infested or not infested with horn flies have been examined. Data have shown significant differences in nitrogen retention, blood cortisol concentrations, vital signs, water consumption, and urine production. Implications are that total energy balance is altered when an animal is exposed to ectoparasite infestations, thereby resulting in decreased productivity.


Sequence of the GLN1 gene of Saccharomyces cerevisiae: role of the upstream region in regulation of glutamine synthetase expression. The GLN1 gene, encoding glutamine synthetase in Saccharomyces cerevisiae, was sequenced, and its encoded polypeptide was shown to have significant homology to other eukaryotic glutamine synthetases. S1 analysis has defined the transcriptional start site of the gene. Upstream analysis of the gene using lacZ fusions has verified transcriptional control of the gene and has identified a nitrogen upstream activation sequence which is required for the increased transcription of GLN1 seen when glutamine is replaced by glutamate as the nitrogen source. cis-acting sites required for the increased transcription in response to purine starvation also have been localized.


Structures of chaperonins from an intracellular symbiont and their functional expression in Escherichia coli groE mutants. An intracellular symbiont harbored by the aphid bacteriocyte, a specialized fat body cell, synthesizes in vivo substantially only one protein, symbionin, which is a member of the chaperonin-60 family of molecular chaperones. Nucleotide sequence determination of the symbionin region of the endosymbiont genome revealed that it contains the two-cistron operon sym. Just like the Escherichia coli groE operon, the sym operon was dually led by a heat shock and an ordinary promoter sequence. According to the nucleotide sequence, symbionin was 85.5% identical to GroEL of E. coli at the amino acid sequence level. SymS, another protein encoded in the sym operon, which is a member of chaperonin-10, was 79.6% identical to GroES. Complementation experiments with E. coli groE mutants showed that the chaperonin-10 and chaperonin-60 genes from the endosymbiont are expressed in E. coli and that they can function as molecular chaperones together with endogenous GroEL and GroES, respectively.


Recent developments in the acute somatic treatment of major depression. The subtypes of major depression are reviewed, and a decision tree for the acute somatic treatment of major depression, including delusional and nondelusional depression, is presented. Considerations in clinicians' decisions about initiation and selection of acute somatic treatment for patients with major depression are then discussed. Current somatic therapies for nondelusional depression are described, with emphasis on the mechanisms of action of the drugs employed, the relationship between mechanism of action and side effect profile, and the selection of a drug on the basis of its side effect profile. The three current strategies for the treatment of delusional depression--electroconvulsive therapy, the combination of an antidepressant and an antipsychotic agent, and amoxapine alone--are then reviewed.


High-performance liquid chromatographic determination of floctafenine and its major metabolite, floctafenic acid in plasma. A simple, rapid and selective high-performance liquid chromatographic method for the determination of floctafenine and its major metabolite, floctafenic acid, in plasma is reported. Plasma samples were purified using the mobile phase as a protein precipitant. The supernatant containing parent compounds and diazepam (internal standard) were eluted from a 5 micron C 18 reversed-phase column at ambient temperature. The mobile phase consisted of 0.05 M sodium acetate:acetonitrile: methanol (200:100:100 v/v/v) adjusted to pH 5 and pumped isocratically at a flow rate of 1.5 ml/min. The effluent was monitored at 350 nm. Quantification was achieved by the measurement of the peak-height ratio of each drug to the internal standard. The mean percentage recoveries from plasma samples spiked with floctafenine and floctafenic acid ranged from 88.13 to 101.93%. Detection limits were 100 ng/ml for floctafenine and 50 ng/ml for floctafenic acid. The coefficients of variation (RSD, %) for within-day and day-to-day analysis were less than 8%.


Complete deletion of the androgen receptor gene: definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status. The molecular basis of androgen insensitivity was investigated in a family with the complete form of the syndrome. Polymerase chain reaction amplification and Southern blot analysis of genomic DNA revealed a deletion of the entire androgen receptor (AR) gene in affected individuals. The carrier status of female members of this family was examined using a HindIII restriction fragment length polymorphism associated with the AR gene. Obligate carriers were hemizygous for one of the two alleles at this locus, while heterozygosity for the polymorphic alleles, implying the presence of two copies of the AR gene, indicated noncarrier status. This conclusion was supported by gene dosage studies using comparative densitometric analysis of Southern blots hybridized simultaneously with an AR cDNA probe and a control cDNA probe from an unrelated gene. Finally, the pattern of inheritance of another X-linked DNA polymorphism allowed us to conclude that the original mutation had occurred in the germ line of the maternal great-grandfather of the index patient. Although rare, complete deletion of the AR gene is of particular importance in terms of correlation between molecular defect and phenotype, as it represents the quintessential form of complete androgen insensitivity, the null phenotype.


The immunohistochemical expressions of epidermal growth factors, epidermal growth factor receptors and c-erbB-2 oncoprotein in human pancreatic cancer. The expressions of epidermal growth factors (EGF), epidermal growth factor receptors (EGFR), and the c-erbB-2 oncoprotein were immunohistochemically examined in 25 cases of human pancreatic carcinoma and epineoplastic pancreatitis and in 10 non-cancerous/non-inflammatory pancreatic tissues. The positive rates of EGF, EGFR, and the c-erbB-2 oncoprotein in cancer tissues were 72%, 36%, and 28%, respectively. EGF was stained mainly in the cytoplasm and partly on the surfaces of the cancer cells. EGFR and the c-erbB-2 oncoprotein were stained mainly on the surfaces of the cancer cells and partly in the cytoplasm. The expressions of these 3 products correlated significantly with tumor invasion into the anterior and posterior areas surrounding the pancreas. In the EGF, EGFR, and c-erbB-2 positive cancer tissues, some stromal cells, that is fibroblasts and endothelial cells, were also positive. In the adjacent pancreatic tissues with inflammation, these products were noted in some ductal cells, acinar cells, fibroblasts and endothelial cells. No distinct staining was detected in non-cancerous/non-inflammatory tissues. The survival period for patients who tested positive for these three proteins was statistically shorter than for those who tested negative. These results suggest that the coexpression of EGF and EGFR and the expression of the c-erbB-2 oncoprotein are related to the existence of the invasion of human pancreatic cancer. Furthermore, an immunohistochemical examination of these three products is useful in forming a prognosis for pancreatic cancer patients.


Modulation of adhesion of lymphocytes to murine brain endothelial cells in vitro: relation to class II major histocompatibility complex expression. Adhesion of lymphocytes to mouse brain endothelial cells was studied after treatment of the endothelium with 1000 U/ml gamma interferon (IFN-gamma) for 1 h to 2 days. Adhesion was not significantly different from controls after 1 h but at 4 h and thereafter, adhesion increased in a time-related manner. IFN-gamma also increased the expression of class II major histocompatibility complex (MHC) and murine intercellular adhesion molecule-1 (ICAM-1) molecules on the endothelial cells. The level of expression of class II MHC molecules was related to the length of exposure to IFN-gamma. MAb blocking studies suggested that class II molecules were responsible for the IFN-gamma-induced increase in lymphocyte-endothelial cell adhesion. Transfection of a murine lung endothelial cell line with cDNA for the class II MHC molecule also produced a significant increase in lymphocyte-endothelial cell adhesion, suggesting that the class II MHC molecule may have a role in adhesion which is distinct from antigen presentation.


Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. A flow cytometric procedure measuring the 5-bromo-2-deoxyuridine incorporated into the DNA of cells in S phase was modified to make it compatible with the immunofluorescence labelling of cell surface antigens. The modifications were introduced at the stages of cell fixation and DNA denaturation. Ethanol was replaced by Tween 20-containing paraformaldehyde and hydrochloric acid was used for the denaturation of DNA by bovine pancreatic DNase-I. These two modifications permitted the preservation of immunofluorescence properties and the use of fluorochromes of the phycobiliprotein family such as phycoerythrin and allophycocyanin. This new procedure is suitable for evaluating leucocyte subsets proliferating in vitro following stimulation. As an illustration the immunosuppressive effect of cyclosporin A on PHA stimulated T4-lymphocytes was evaluated.


Perfusion of immobilized isolated nerve terminals as a model for the regulation of transmitter release: release of different, endogenous transmitters, repeated stimulation, and high time resolution. To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (greater than 30 min) inside the chamber and did not elute from the chamber, which allowed long-term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca(2+)-dependent (exocytotic) and Ca(2+)-independent manner; (c) a step-like stimulation with depolarizing agents (rise time, 3-4 s) and a high time resolution (600-ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus-secretion coupling, co-transmission, and the exo-endocytosis cycle.


Effect of histamine H2 receptor antagonists on the secretion of cerebrospinal fluid in the cat. Following a recent report that epithelial cells of the choroid plexus possess histamine H2 receptors, the effect of cimetidine and ranitidine, histamine H2 receptor antagonists, on the secretion and electrolyte content of CSF was examined. Fifty cats were divided into one control (n = 6) and six experimental groups. CSF was collected by puncture of the cisterna magna following pentobarbital anesthesia, and its volume, concentrations of Na+, K+, Cl-, and pH were determined. Cimetidine or ranitidine (50, 20, or 10 mg/kg) was injected intravenously 2 h after the start of the test, and their concentrations were measured in hourly blood samples and in 30-min aliquots of CSF in the 50 mg/kg experimental groups. Whereas the secretion of CSF did not change over 6 h in the control group, it decreased significantly by 30-60 min after injection of cimetidine or ranitidine and remained low for the following 6 1/2 h in all experimental groups except the 10-mg ranitidine group. Peak cimetidine and ranitidine concentrations in CSF in the 50-mg experimental groups were noted 60 and 90 min, respectively, after intravenous injection. CSF electrolyte concentrations and pH did not change during the test in any group. We conclude that intravenous cimetidine or ranitidine can significantly reduce CSF secretion in the cat, possibly by competitive inhibition of the histamine effect on H2 receptors located on the choroid plexus epithelial cell, or by a direct effect on the capillaries of the choroid plexus.


D1 dopamine receptor activation of multiple transcription factor genes in rat striatum. Recent studies have shown that dopamine receptor agonists induce expression of Fos-like immunoreactivity in rat striatal neurons. The protooncogene c-fos belongs to a family of immediate early genes that are rapidly induced in fibroblasts by growth factors. In light of previous findings that several immediate early gene mRNAs that encode proven or putative transcription factors are differentially regulated by neuronal stimulation in vivo, we have examined the effect of dopaminergic agents on mRNA levels of several such genes using in situ hybridization and northern blot analysis. d-Amphetamine (2.5-10 mg/kg i.p.) causes a rapid but transient dose-dependent increase in zif268 and jun-B mRNA levels in striatum that was abolished by striatal 6-hydroxydopamine lesions or by pretreatment with the specific D1 receptor antagonist SCH-23390 but not by specific D2 receptor antagonists. Apomorphine, a dopamine agonist that acts at both D1 and D2 receptors, and SKF-38393, a specific D1 receptor agonist, produce similar mRNA changes in rats pretreated with either 6-hydroxydopamine or reserpine, whereas LY-171,555, a specific D2 receptor agonist, has no effect. Direct dopamine agonist effects on these immediate early gene mRNA levels are also blocked by D1 but not by D2 antagonists. We observed similar, although less robust, changes in c-fos and fos-B mRNA levels. These results demonstrate that striatal D1 dopamine receptors are coupled to activation of multiple transcription factor genes, including zif268 and jun-B as well as members of the fos family.


Clonidine inhibition of norepinephrine release from normal and morphine-tolerant guinea pig cortical slices. Endogenous norepinephrine (NE) release in cerebral cortex slices taken from normal and morphine-tolerant guinea pigs was measured by HPLC. In normal slices, a linear relationship was found between electrically evoked NE release and the log of the frequency of stimulation in the range of 1-20 Hz. The efficiency of the alpha 2-mediated autofeedback was tested by adding the alpha 2-agonist clonidine and the alpha 2 agonist idazoxan. NE release was dose-dependently reduced by clonidine (1 nmol/L-1 mumol/L) and increased by idazoxan (10-100 nmol/L). The inhibition by clonidine was significantly greater at 1 Hz than at 3 Hz, whereas the absolute increase in NE release induced by idazoxan was greater at 3 Hz than at 1 Hz. Morphine at 1 mumol/L (a concentration per se ineffective) shifted to the left the clonidine concentrations able to inhibit NE release at 3 and 1 Hz (1-10 nmol/L), but at both frequencies, the opiate reduced the maximal inhibition induced by clonidine at 1 mumol/L. In slices taken from morphine-tolerant guinea pigs (in the presence of morphine at 1 mumol/L), clonidine (1 nmol/L-1 mumol/L) was ineffective at the stimulation rate of 3 Hz, but it was more active than in normal slices at 1 Hz. Such a response pattern suggests a reduced availability of alpha 2 receptors and an increase in their sensitivity to clonidine. However, chronic morphine treatment did not influence the physiological autoinhibition because the increase in NE release elicited by idazoxan (10-100 nmol/L) at 1 and 3 Hz was the same in normal and in "morphine-tolerant" slices.(ABSTRACT TRUNCATED AT 250 WORDS)


Putative neurotoxicity of SKF 38393 and other D1 dopaminergic drugs investigated in rat striatum. The recently alleged neurotoxicity of the D1 receptor agonist, SKF 38393, was investigated in rat striatum by measuring the enzymes acetylcholinesterase (AChE) and glutamate decarboxylase (GAD). First, unilateral intrastriatal microinjection of the excitotoxin kainic acid (2 micrograms in 1 microliter) was shown to evoke vigorous contraversive circling, followed 1 or 2 weeks later by profound decreases in striatal AChE (24 and 54%), GAD (51 and 75%), and protein (36 and 47%), as well as loss of GAD (45% at 2 weeks) in the ipsilateral substantia nigra. Similar striatal treatments with SKF 38393 (30 micrograms in 0.5-1 microliter), the related benzazepines SKF 82526 (D1 agonist, 30 micrograms in 1 microliter) and SCH 23390 (D1 antagonist, 5 micrograms in 1 microliter), or the phenanthridine D1 agonist CY 208-243 (5 micrograms in 1 microliter) failed to affect the rats' behaviour or their striatal levels of AChE, GAD, and protein. Intrastriatal SKF 38393 (30 micrograms in 0.5 microliter) also had no influence on these enzymes in the substantia nigra. It is concluded that none of the D1 dopaminergic compounds examined here was neurotoxic toward the many different cell groups that contain AChE and/or GAD in the striatum.


The unc-18 gene encodes a novel protein affecting the kinetics of acetylcholine metabolism in the nematode Caenorhabditis elegans. Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc-18. We isolated a transposon-insertion allele for unc-18 and used it to clone a genomic region containing the unc-18 locus. The unc-18 location within this region was determined by rescuing the unc-18 mutant phenotype in a germ-line transformation experiment and identifying transcripts affected by four independent unc-18 mutations. A single-sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc-18 gene plays a role in development as well as in the kinetics of ACh metabolism.


Regulation of tyrosine hydroxylase gene expression in the rat carotid body by hypoxia. The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.


Regional distribution of alpha 2A- and alpha 2B-adrenoceptor subtypes in postmortem human brain. The newly available and highly selective radiolabeled antagonist [3H]RX 821002 was used to examine the distribution of alpha 2 adrenoceptors in human brain. High densities of alpha 2 adrenoceptors were found in the hippocampus, frontal cortex, thalamus, amygdala, pons, and medulla oblongata. Intermediate densities were observed in the striatum (nucleus accumbens, nucleus caudatus, and putamen), globus pallidus, and substantia nigra. The KD values for [3H]RX 821002 were similar in all regions (ranging from 2.8 to 7.5 nM). On the basis of their different affinities for prazosin and oxymetazoline, the alpha 2 adrenoceptors have been divided into alpha 2A and alpha 2B subtypes. To examine the alpha 2A/alpha 2B-adrenoceptor ratio in the different brain regions, we performed oxymetazoline and prazosin/[3H]RX 821002 competition binding experiments. In frontal cortex membranes, the competition curves with prazosin were steep, indicating a single class of binding sites, whereas the competition curves with oxymetazoline were shallow and fitted by computer best to a two-site model. However, in the presence of GTP, the high-affinity sites for oxymetazoline were partially converted into low-affinity sites, indicating that this agonist interacts with high- and low-affinity states of the alpha 2 adrenoceptors. This implies that oxymetazoline is not very suitable for discriminating the alpha 2A- and alpha 2B-receptor subtypes in radioligand binding studies. Therefore, prazosin/[3H]RX 821002 competition binding experiments were used to investigate the distribution of the alpha 2-adrenoceptor subtypes in human brain. The alpha 2A-receptor subtype was detected in all brain regions examined. In contrast, alpha 2B receptors were only observed in striatum and globus pallidus.


Comparison by race of total serum IgG, IgA, and IgM with CD4+ T-cell counts in North American persons infected with the human immunodeficiency virus type 1. European patients with human immunodeficiency virus type 1 (HIV-1) infection have been reported to have lower titers of anti-p24 antibody than Central African HIV seropositive patients. Recently, black HIV positive patients in the United States were reported to be more likely to have detectable anti-p24 antibodies, less p24 antigenemia, and higher combined serum immunoglobulins than white HIV positive patients. We measured individual total serum immunoglobulins in 853 HIV positive patients (94% male; 58% white and 42% black) on their initial medical evaluation and compared them with CD4+ T-cell counts. Blacks had notably higher IgG levels (p = 0.001) across the entire spectrum of CD4+ T-cell counts. Serum IgM levels were slightly higher in blacks. IgA levels were not significantly different between the races, although the trend (p = 0.006) was toward higher levels in whites. We also measured these three serum immunoglobulins in 60 HIV seronegative, healthy blood donors (30 black and 30 white). In this control group, blacks had statistically higher IgG and IgA levels than whites. A review of the literature prior to the HIV/acquired immune deficiency syndrome epidemic also supports the view that racial differences in IgG levels are not specific for HIV infection. We speculate that racial differences in humoral immunity, independent of geography or strain of HIV, may account for differences in anti-HIV antibody levels and HIV antigenemia.


DNA polymorphisms of the human CD46 gene in fetal and adult tissues. DNA polymorphisms of the CD46 gene were investigated using a full length cDNA probe and five different restriction enzymes in genomic DNA samples from human fetal tissues and adult peripheral blood leukocytes. A HindIII polymorphism was observed and was classed as Types I, II and III at a ratio of approximately 3.5:2:1, depending on the presence or absence of two polymorphic fragments of 4.5 and 2.0 kb. No variation of polymorphic distribution was evident between the fetal and adult samples. Whether variation in CD46 expression between maternal and fetal tissue is relevant to fetal viability in pregnancy remains to be established.


Small atrial natriuretic peptide analogues: design, synthesis, and structural requirements for guanylate cyclase activation. Structure/activity studies on atrial natriuretic peptide ANP (1-28) have highlighted three portions of the native molecule as necessary for its biological responses. We have linked these three regions and excised the remaining segments to produce a family of small analogues (less than half the size of the parent) which demonstrate the full range of ANP's actions. Importantly, these compounds act at both major types of ANP receptor. Two critical modifications lead to more potent analogues; both involve expanding the cyclic portion of the molecule. Further optimization of one of these modified structures leads to A68828, a full ANP agonist which shows promise as a preventative agent against acute renal failure.


Analgesic dipeptide derivatives. 7. 3,7-Diamino-2-hydroxyheptanoic acid (DAHHA) containing dipeptide analogues of the analgesic compound H-Lys-Trp(Nps)-OMe. A series of diastereomeric dipeptides, analogues of the analgesic compound H-Lys-Trp(Nps)-OMe (2), containing 3,7-diamino-2-hydroxyheptanoic acid (DAHHA) and 2-[(o-nitrophenyl)sulfenyl]tryptophan [Trp(Nps)] has been synthesized. These compounds were tested as enkephalin-degrading aminopeptidases (APs), AP-M and AP-B inhibitors, and analgesics. The inhibitory potencies and the antinociceptive effects depended on the stereochemistry of the compounds. (2S,3R)-DAHHA-L-Trp(Nps)-OMe (26d) was a highly potent and selective enkephalin-degrading APs inhibitor, with an IC50 value in the 10(-8) M range. Although this derivative was about 10(3)-fold more potent than 2 against these enzymes, their antinociceptive effects were completely similar. These results indicate that the inhibitory capacity of this series of Trp(Nps)-containing dipeptides against enkephalin-degrading enzymes is not an important factor for their antinociceptive effects.


Choosing between apples and apples: physicians' choices of prescription drugs that have similar side effects and efficacies. To examine physician choices of commonly used medications having similar side effects and efficacies, and to evaluate factors that may affect these choices. Cross-sectional survey conducted in winter 1989-1990. 263 physicians at a university teaching hospital (response rate = 71%). Physicians rated patient compliance, cost to patient, and patient preference as the three most influential factors in their selection of a particular agent from a class of similar drugs. Housestaff were less likely than faculty to consider cost to patient as a "very important" factor (33% vs. 60%; p less than 0.05), and only 11% of all physicians felt that cost to third-party payer was very important. Physicians reported that their choices of particular nonsteroidal anti-inflammatory drugs (NSAIDs), histamine-2 (H2) blockers, and inhaled beta-agonists were mainly determined by which drugs enhanced compliance or were used by others (the "traditional choice"); cost to patient was a less important influence in these instances. All physician subgroups were inaccurate in predicting the approximate prices of their first- and second-choice agents. For example, only 28% of those selecting naproxen as their preferred NSAID were within $10 of the range of the prices of a one-month supply, and 14% were within $10 for cimetidine. Although this group of physicians reported considering drug costs to be important when choosing between similar drugs, they acknowledged that cost was relatively unimportant in several specific instances studied and their knowledge of the absolute and relative prices of drugs they commonly prescribed was deficient.


Use of contour-clamped homogeneous electric field (CHEF) electrophoresis to type methicillin-resistant Staphylococcus aureus. Strains of methicillin-resistant Staphylococcus aureus (MRSA) from Australia and the UK were compared by digesting their chromosomal DNA with the low-frequency-cutting restriction enzyme SmaI and separating the restriction fragment length polymorphisms (RFLPs) by contour-clamped homogeneous electric field (CHEF) electrophoresis. The numbers of restriction fragments produced were in the range 14-17 and the sizes of the bands were 7-700 kb. Generally, the results confirmed previous conclusions based on antimicrobial resistance and plasmid profiles. The earlier MRSA isolates were different from more recent isolates, and the epidemic MRSA from eastern Australia (EA MRSA) was the same as the epidemic MRSA (EMRSA) found in London hospitals. However, contrary to previous results, the EA MRSA did not constitute a homogeneous group. The results showed that comparison of RFLPs by CHEF electrophoresis is a useful technique for studying the epidemiology of MRSA.


The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Interferon resistance of vaccinia virus is mediated by specific inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by the double-stranded-RNA-activated (DAI) protein kinase. Vaccinia virus encodes a homolog of eIF-2 alpha, K3L, the deletion of which renders the virus sensitive to interferon treatment. We have studied the mechanism by which this protein product elicits interferon resistance in a transient DNA transfection system designed to evaluate regulators of eIF-2 alpha phosphorylation. In this system, translation of a reporter gene mRNA is inefficient because of eIF-2 phosphorylation mediated by the DAI protein kinase. Cotransfection of the K3L gene enhances translation of the reporter mRNA in this system. The K3L protein inhibits eIF-2 alpha phosphorylation and DAI kinase activation, apparently without being phosphorylated itself. Inhibition of protein synthesis, elicited by expression of a mutant Ser-51----Asp eIF-2 alpha designed to mimic a phosphorylated serine, is not relieved by the presence of K3L, suggesting that K3L cannot bypass a block imposed by eIF-2 alpha phosphorylation. The results suggest that K3L acts as a decoy of eIF-2 alpha to inhibit DAI kinase autophosphorylation and activation. Another vaccinia virus gene product, K1L, which is required for growth of vaccinia virus on human cells, does not enhance translation in this assay.


Stimulation of lymphokines in Jurkat cells persistently infected with vaccinia virus. The response of the human CD4+ T-cell line Jurkat to infection with vaccinia virus was investigated. Virus titers peaked approximately 3 to 4 days after infection, while cell growth paralleled that of uninfected cells, indicating that growth rates were not appreciably affected by viral infection. Results from plaque assays and fluorescence-activated cell sorter (FACS) analyses of virus antigens demonstrated that a persistent infection in which the percentage of infected cells and the virus titers fluctuated from passage to passage was established. Further characterization of the persistent infection revealed that the virus influences cellular functions. Induction of interleukin-2 (IL-2) and IL-2 receptor alpha (IL-2R alpha) in Jvac cells was shown by enzyme-linked immunosorbent assay and FACS analysis, respectively. Hybridization of cellular RNA with cloned probes confirmed the increased IL-2 expression and demonstrated that Jvac cells also expressed more IL-6 but not gamma interferon (IFN-gamma) or IL-1 beta. Dual-antibody staining and FACS analysis for vaccinia virus antigens and IL-2R alpha indicated that IL-2R alpha expression was restricted to the infected cells. Jvac cells were also resistant to superinfection, an additional proof that persistent infection elicited phenotypic changes in the cell population.


Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent. Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.


Multiple isolates and characteristics of human T-cell leukemia virus type II. Human T-cell leukemia (or lymphotropic) virus type II (HTLV-II) was isolated from eight HTLV-seropositive patients, six of whom were also infected with human immunodeficiency virus, by cocultivation of peripheral blood mononuclear cells (PBMCs) with BJAB, a continuous B-cell line. Restriction endonuclease mapping of the proviruses demonstrated consistent differences among isolates, and two distinct physical map patterns were observed. The results suggest the existence of two closely related molecular subtypes of HTLV-II, which are tentatively designated HTLV-IIa and HTLV-IIb. This finding was supported by preliminary nucleotide sequence analysis of the env gene region encoding the transmembrane glycoprotein gp21, which showed consistent differences between the two proposed virus subtypes. Exploitation of differences in restriction endonuclease sites allowed polymerase chain reaction amplification to detect and differentiate the two subtypes in fresh PBMCs of HTLV-seropositive intravenous drug abusers (IVDAs). The results of these studies confirm that HTLV-II infection is the prominent HTLV infection in seropositive IVDAs and also show that infection with both subtypes occurs. The finding of genetic heterogeneity in the HTLV-II group of viruses may have important implications for studies on its role in human disease and will be useful in characterizing the viruses present in newly discovered endemic foci in New World indigenous populations.


Differential effects of LHRH and somatostatin analogs on human breast cancer. We have been interested in the possible direct effects of luteinizing hormone releasing hormone (LHRH) and somatostatin (SS) analogs on the growth of human mammary tumor cells. Four recently synthesized peptide hormones including the LHRH agonists D-Trp6-LHRH and zoladex, LHRH antagonists SB30 and SB75, and the somatostatin analog RC 160 were analyzed for their effects on DNA synthesis of MCF-7 breast cancer cells in culture. At 48 hr, D-Trp6-LHRH and SB30 did not show significant effects (dose range, 10(-12)-10(-6) M). However, the combination of these two peptides at 10(-10) M produced significant inhibition of 3[H]thymidine incorporation (50% control). At 72 hr in the absence of estradiol-stimulated growth, D-Trp6-LHRH showed inhibition at 10(-12) and 10(-10) M (P less than 0.005 and 0.001). At higher concentrations, no significant inhibition was noted. In contrast to D-Trp6, SB30 (antagonist) showed no inhibition but significant stimulation of DNA synthesis at 10(-6) and 10(-4) M. In the presence of added estradiol (10(-9) M), complete reversal of D-Trp6-LHRH analog inhibition is noted. In contrast, there is persistent stimulation by SB30 (P less than 0.001). At 96 hr, D-Trp6-LHRH continued to show maximal inhibition of 70% in the absence of estradiol. SB30 stimulated DNA synthesis 100% at 10(-6) M. At 72 hr, the SS analog RC 160 demonstrated significant inhibition (53%) that was similar to D-Trp6 and SB75 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160. The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.


Immunoglobulin prophylaxis against HTLV-I in a rabbit model. We have investigated the protective effect of human T-cell leukemia virus I (HTLV-I) immune globulin (HTLVIG) against HTLV-I in rabbits. HTLVIG containing 77 mg/ml of IgG was prepared from pooled plasma from seropositive healthy persons. In the first experiment, four groups (A, B, C, and D) of three rabbits were transfused with 5 ml blood from an HTLV-I-infected rabbit. Groups A, B, and C were infused 24 h later with 10, 5, and 2 ml HTLVIG, respectively, while group D was infused with 10 ml HTLVIG 48 h later. Seroconversion for HTLV-I occurred in none of group A, one of group B, and all of groups C and D after 2-5 weeks. In the second experiment, four litters (E, F, G, and H) born to another virus-infected rabbit and consisting of 7, 5, 7, and 7 newborns, respectively, were used. Litters E and H were allowed to grow normally as controls, while litters F and G were given intraperitoneal inoculation of 3 ml/kg of HTLVIG weekly four times until weaning. Although three of litters E and H each seroconverted after 5-8 weeks, none of litters F, and one of litter G became antibody-positive after 10 weeks. Presence or absence of HTLV-I infection in all these animals was confirmed by transfusion assay or gene amplification. These results indicate that passive immunization protects rabbits against blood- and milk-borne transmission of HTLV-I.


ISIS-3: a randomised comparison of streptokinase vs tissue plasminogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41,299 cases of suspected acute myocardial infarction. ISIS-3 (Third International Study of Infarct Survival) Collaborative Group. 41,299 patients entering 914 hospitals up to 24 h (median 4 h) after the onset of suspected acute myocardial infarction were randomised between streptokinase (SK: 1.5 MU infused over about 1 h), tissue plasminogen activator (tPA, duteplase: 0.60 MU/kg infused over about 4 h), or anisoylated plasminogen-streptokinase activator complex (APSAC), anistreplase: 30 U over about 3 min). All patients were to receive aspirin (162 mg/day enteric-coated), with the first tablet chewed for rapid and full antiplatelet effect. Half of all patients were randomly allocated subcutaneous calcium heparin (12,500 IU starting at 4 h and given twice daily for 7 days or until prior discharge) in addition to aspirin, and the other half were to receive aspirin alone. ASPIRIN PLUS HEPARIN VERSUS ASPIRIN ALONE--The addition of heparin to aspirin was associated with an excess of transfused or other major non-cerebral bleeds (1.0% aspirin plus heparin vs 0.8% aspirin alone; 2p < 0.01) and of definite or probable cerebral haemorrhage (0.56% vs 0.40%; 2p < 0.05), but with no significnat differences in total stroke (1.28% vs 1.18%). Reinfarctions were slightly less common among those allocated aspirin plus heparin (3.16% vs 3.47%; 2p = 0.09). There was no signficant difference in the pre-specified endpoint of 35-day mortality (2132 [10.3%] aspirin plus heparin vs 2189 [10.6%] aspirin alone). During the scheduled heparin treatment period there were slightly fewer deaths in the aspirin plus heparin group (days 0-7 in hospital: 1534 [7.4%] vs 1633 [7.9%]; 2 p = 0.06), with a slight convergence by day 35 (598 further deaths [3.1% of survivors] vs 556 [2.9%]). The pattern was similar to that observed in the GISSI-2 trial, so that in both trials combined there was a significant reduction in mortality during the scheduled treatment period (2071 [6.8%] vs 2239 [7.3%]; 2p < 0.01). This indicates avoidance of 5 deaths (SD 2) per 1000 patients allocated this high-dose subcutaneous heparin regimen in addition to aspirin, but some of any early benefit may be lost after heparin ceases, with no significant mortality advantage in days 0-35 (both trials: 3100 [10.0%] vs 3172 [10.2%]) or during follow-up to 6 months. SK VERSUS APSAC--APSAC was associated with significantly more reports of allergy causing persistent symptoms and of non-cerebral bleeds, but not of transfused bleeds or of reinfarctions. There was a slight excess of strokes with APSAC (1.04% SK vs 1.26% APSAC; 2p = 0.08), much of it appearing soon after treatment started (strokes during days 0-1: 0.50% SK vs 0.73% APSAC; 2p < 0.02) and being attributed to cerebral haemorrhage (0.24% SK vs 0.55% APSAC; 2p < 0.0001). No significant difference was observed in reinfarction (3.47% SK vs 3.55% APSAC). There was no significant mortality difference during days 0-35, either among all randomised patients (1455 [10.6%] SK vs 1448 [10.5%] APSAC) or among the pre-specified subset presenting within 0-6 h of pain onset and with ST elevation on the electrocardiogram in whom fibrinolytic treatment may have most to offer (861 [10.0%] SK vs 855 [9.9%] APSAC). No significant difference in 6-month survival was apparent overall or in the subset.(ABSTRACT TRUNCATED AT 400 WORDS)


Identification by molecular cloning of an autoantigen associated with Addison's disease as steroid 17 alpha-hydroxylase. Idiopathic Addison's disease is characterised by a progressive failure in the synthesis of all classes of steroid hormones and by an immune response against the steroid-producing cells of the adrenal cortex; the nature of the adrenal autoantigens is not known. We have used molecular cloning and sequencing to identify the target antigens. We screened a human fetal adrenal cDNA expression library in lambda gt11 vector with serum samples from patients with Addison's disease as part of the type 1 polyendocrine autoimmunity syndrome. Samples from 3 patients, which had precipitating antibodies against two adrenal proteins detected by immunodiffusion and against five adrenal proteins of molecular mass 55, 48, 43, 39, and 19 kDa as judged by immunoblotting, were used to identify 60 immunoreactive clones. 39 of these were subcloned, inserted into the M13mp10 vector, and sequenced by the dideoxy method or identified by Southern and dot-blot hybridisation. All but 1 of the inserts showed more than 98.8% homology with the published sequence of steroid 17 alpha-hydroxylase. This protein was expressed by insertion of 1 of the clones into the pGEMEX-1 vector. Only serum from patients with Addison's disease and type 1 polyendocrine autoimmunity syndrome that reacted with the 55 kDa adrenal protein recognised the recombinant 17 alpha-hydroxylase protein on immunoblotting. Our results show that one of the key enzymes in steroid biosynthesis, 17 alpha-hydroxylase, is an autoantigen involved in the pathogenesis of adrenocortical failure.


Recombinant human erythropoietin for autologous blood donation: effects on perioperative red-blood-cell and serum erythropoietin production. To speed collection of blood for autologous transfusion during elective surgery, patients may be given recombinant human erythropoietin (r-HuEPO). In a controlled trial, we evaluated the effects of r-HuEPO on perioperative red-blood-cell and serum erythropoietin (s-EPO) production in patients donating blood before elective orthopaedic surgery. Patients were assigned randomly to receive no r-HuEPO (12 patients), or 3000 U (4), 6000 U (5), or 9000 U (4) of r-HuEPO intravenously twice a week from the time of the first blood donation. All patients received iron sulphate. 1200 ml blood was collected from each patient in three weekly donations of 400 ml. The 3000, 6000, and 9000 U treatment groups produced 284, 350, and 383 ml, respectively, of red cells during donation, and the untreated controls produced 211 ml. s-EPO concentrations were within the normal range during donation. After surgery, s-EPO concentrations peaked on postoperative day 1 in untreated patients and on day 7 in treated patients; therefore, r-HuEPO may suppress endogenous erythropoietin secretion. Although administration of r-HuEPO increases production of red blood cells, the preoperative anaemia induced by repeated phlebotomy without r-HuEPO may accelerate the postoperative secretion of endogenous erythropoietin.


Choriodecidual production of interleukin-8 and mechanism of parturition. Both prostaglandins and antiprogestagens can induce labour and ripen the cervix, but the mechanisms are unclear. The collagenases that bring about cervical ripening are neutrophil derived. We examined the potential of uterine tissues to control neutrophil attraction by measuring interleukin-8 production. Choriodecidual cells in culture produced substantial amounts of interleukin-8; release was inhibited by progesterone and stimulated by the antiprogestagen mifepristone. Interleukin-8 production was similar in cells from spontaneously delivered placentas and from those obtained at caesarean section. Since prostaglandin E and interleukin-8 have synergistic effects, we suggest that interleukin-8 activity is the final common step of prostaglandin and antiprogestagen action in parturition.


Induction of thromboses within renal grafts by high-dose prophylactic OKT3. Among 93 consecutive kidney-transplant patients who received prophylactic OKT3 10 mg/day for 2 weeks, 9 had intragraft thromboses within 2 weeks of transplantation. The thromboses were in graft artery in 1 patient and veins in 3. The other 5 had thromboses in glomerular capillaries and thrombotic microangiopathy similar to that of haemolytic-uraemic syndrome. All attempted treatments failed, and the 9 grafts had to be removed. The finding that plasma concentrations of prothrombin fragment 1 and 2 were higher 4 h after the first OKT3 dose in OKT3 recipients than in transplant patients who received other prophylaxis (mean 5.88 [SEM 0.76] vs 2.25 [0.59] nmol/l, p less than 0.01) confirms that OKT3 has procoagulant effects in vivo.


Intrauterine devices and pelvic inflammatory disease: an international perspective. The risk of pelvic inflammatory disease (PID) associated with use of an intrauterine device (IUD) has been an important concern that has dominated decisions on its use throughout the world, especially in the USA. Early research that suggested such an association led to both a dramatic decline in use of the method and its withdrawal from the US market by two manufacturers. However, factors other than use of an IUD are now thought to be major determinants of PID risk. To address these concerns, we have reviewed the World Health Organisation's IUD clinical trial data to explore the incidence and patterns of PID risk with use of an IUD. The overall rate of PID among 22,908 IUD insertions and during 51,399 woman-years of follow-up was 1.6 cases per 1000 woman-years of use. After adjustment for confounding factors, PID risk was more than six times higher during the 20 days after insertion than during later times (unadjusted rates, 9.7 vs 1.4 per 1000 woman-years, respectively); the risk was low and constant for up to eight years of follow-up. Rates varied according to geographical area (highest in Africa and lowest in China) and were inversely associated with age. PID rates were lower among women who had IUDs inserted more recently. Our findings indicate that PID among IUD users is most strongly related to the insertion process and to background risk of sexually transmissible disease. PID is an infrequent event beyond the first 20 days after insertion. Because of this increased risk with insertion, IUDs should be left in place up to their maximum lifespan and should not routinely be replaced earlier, provided there are no contraindications to continued use and the woman wishes to continue with the device.


Magnetic resonance angiography of renal transplants. Stenosis of the artery of transplanted kidneys is an important cause of graft dysfunction. Diagnosis and follow-up of this condition normally requires intra-arterial digital-subtraction angiography (IADSA), which is invasive and may cause complications. A possible alternative to IADSA is magnetic resonance angiography (MRA), and we have assessed this technique in 50 renal transplant patients who were referred for investigation of possible renal arterial stenosis. In every patient, MRA was compared prospectively with conventional IADSA. Compared with IADSA, MRA had a sensitivity of 83% and a specificity of 97%, and when all images were graded retrospectively for severity of stenosis, the two techniques showed a significant correlation (r = 0.74, p less than 0.001). MRA can provide an accurate image of the renal transplant artery in a non-invasive manner with a high sensitivity and specificity.


Randomised trial of artesunate and mefloquine alone and in sequence for acute uncomplicated falciparum malaria. The increasing frequency of therapeutic failures in falciparum malaria in Thailand shows an urgent need for effective drugs or drug combinations. Artesunate, a qinghaosu derivative, is effective in clearing parasitaemia rapidly, but the recrudescence rate can be as high as 50%. We have compared artesunate followed by mefloquine with each drug alone in acute, uncomplicated falciparum malaria. 127 patients were randomly assigned treatment with artesunate (600 mg over 5 days), mefloquine (750 mg then 500 mg 6 h later), or artesunate followed by mefloquine. All patients were admitted to hospital for 28 days to exclude reinfection. Cure was defined as no recrudescence during the 28 days' follow-up. The cure rates for mefloquine and artesunate alone were 81% (30/37 patients) and 88% (35/40); the combination was effective in all of 39 patients. Fever and parasite clearance times were significantly shorter in the groups that received artesunate than in the mefloquine-only group. The frequency of nausea and vomiting was slightly, but not significantly, higher among patients who received both drugs than in the other groups. The combination of artesunate followed by mefloquine is highly effective and well tolerated in patients with acute, uncomplicated falciparum malaria in Thailand.


T-cell receptor variable gene products and early HIV-1 infection. To assess the hypothesis that the human immunodeficiency virus (HIV) might mimic major histocompatibility complex (MHC) allodeterminants and interact with T-cell receptors (TCRs) of alloreactive T-cells, we have done a preliminary analysis of the range of alpha beta TCR gene products in 16 HIV-1-seropositive individuals with normal CD4 counts and in 16 healthy HIV-1-negative controls. Using a panel of monoclonal antibodies with a two-colour direct immunofluorescence method, we found a significant increase in the expression of the V beta 5.3 subfamily in the HIV-positive patient group compared with controls (p less than 0.01). Selected increase in expression of V beta sequences has been described in various autoimmune conditions and our findings raise the possibility that the immunopathological damage from HIV infection may be due to the induction of autoreactivity. If HIV does mimic MHC II, the normal immune response to the virus could represent an autoimmune process similar to graft-versus-host disease.


Conservative treatment of mild/moderate cervical dyskaryosis: long-term outcome. There is some controversy about the management of women with mild or moderate dyskaryotic cervical smears. To assess the strategy of an established cervical cytology screening programme (Grampian region, northeast Scotland) we identified 500 women who had had mild or moderate cervical dyskaryosis in 1978 or 1979, and 500, matched by age, who had had a normal smear at that time. Follow-up smear results and any subsequent investigation by colposcopy, cone biopsy, or hysterectomy, with biopsy result were recorded. Of the 500 women who initially had an abnormal smear, 300 (60%) had a smear that was normal or inflammatory at their last visit (after seven years' median follow-up). 184 (37%) had undergone biopsy, 97 (19%) of whom were cervical intraepithelial neoplasia grade III or worse. Survival curves for time to biopsy and ten-year biopsy rates show that women with an abnormal smear before their baseline year were the most likely to have a subsequent biopsy. Older women had a biopsy less often and at biopsy were more likely to have minor abnormalities. Mild or moderate dyskaryotic smears should not be an indication for immediate referral for colposcopy, since under a conservative management policy most women return to normal without needing treatment. Nevertheless, the increased risk associated with abnormal smears justifies rigorous surveillance.


Effect of calcitonin-gene-related peptide in patients with delayed postoperative cerebral ischaemia after aneurysmal subarachnoid haemorrhage. European CGRP in Subarachnoid Haemorrhage Study Group. The finding that the carotid vascular beds are sensitive to the potent vasodilator calcitonin-gene-related peptide (CGRP) suggested that the drug might help to prevent ischaemic deterioration after surgery for aneurysmal subarachnoid haemorrhage (SAH). The results of a preliminary study were encouraging, so we have carried out a randomised multicentre single-blind comparison of CGRP and standard best management in patients with ischaemic deficits after surgery for ruptured intracranial aneurysms. Patients aged 18-70 years in whom a focal neurological deficit developed or who had a reduction of 2 or more points on the Glasgow coma scale (GCS) after surgery entered the study after computed tomography had excluded non-ischaemic causes for the neurological deficit. 62 patients were randomly assigned an infusion of 0.6 micrograms/min CGRP for 4 h, then up to a maximum of 10 days, and 55 patients standard best management (controls). GCS and haemodynamic variables were assessed during the hospital stay, and all patients were followed up at 3 months by an independent investigator, who was unaware of their treatment. Outcome, measured on the Glasgow outcome scale, at 3 months was good in 66% of those treated with CGRP and 60% in the controls; the relative risk of a poor outcome in CGRP-treated patients was 0.88 (95% confidence interval 0.60 to 1.28). Hypotension was a common side-effect of the CGRP infusion. 66% of the CGRP group did not complete treatment because of adverse events (19 patients), lack of improvement at 4 h (17 patients) or later (4 patients), or patient's request (1 patient). Although we could not show a significant beneficial effect of CGRP in this trial, the wide confidence interval for the risk of a poor outcome and the fact that only a third of patients completed treatment mean that a clinically useful benefit cannot yet be ruled out.


High incidence of primary gastric lymphoma in northeastern Italy. We previously noted an extraordinarily high number of cases of primary gastric lymphoma (PGL) in northeastern Italy. We have now formally compared the incidence in Feltre, Italy, with that in three similar communities in the UK. Each community has a stable population served by a single endoscopy unit and histopathology laboratory. There were 13 times more cases of PGL in Feltre in 1986-91 than in the UK communities (66 vs 5 per 100,000 per 5 years). The incidence of gastric adenocarcinoma was also substantially higher in Feltre than in the UK (270 vs an average of 82 per 100,000 per 5 years), as was the prevalence of gastritis associated with Helicobacter pylori infection (87% of 1343 gastric biopsy samples in 1991).


Urinary excretion of platelet-activating factor in haemolytic uraemic syndrome. Since some of the features of haemolytic uraemic syndrome (HUS), such as platelet activation and glomerular injury, could be brought about by platelet-activating factor (PAF), we have studied the urinary excretion of PAF in 10 children with HUS and in 10 healthy age-matched controls. Urinary PAF concentrations, measured by radioimmunoassay, were significantly higher in acute-phase HUS patients than in controls (mean 2.04 [SD 1.66] vs 0.72 [0.43] ng/mg creatinine, p less than 0.05) but were similar to those in controls in samples taken after recovery. High PAF concentrations during the acute phase of HUS may reflect platelet activation and glomerular injury; the lower values after recovery suggest that urinary PAF may be a marker of disease activity.


Effects of increased inspired oxygen concentrations on exercise performance in chronic heart failure. Exercise capacity in patients with stable heart failure may be influenced by prolonged drug treatment or exercise training, but acute interventions are generally thought to have little effect. Cardiorespiratory responses to exercise were studied in 12 consecutive patients with chronic congestive heart failure who underwent serial submaximal and maximal exercise tests at inspired oxygen concentrations of 21% (room air), 30%, and 50%. Mean (SD) exercise duration during progressive testing to maximum exercise capacity was prolonged from 548 (276) s on room air to 632 (285) s on 50% oxygen (p = 0.012). During steady-state exercise at 45 W, oxygen enrichment to 50% was associated with significantly increased arterial oxygen saturation (94.6 [1.9]% to 97.5 [1.3]%), and significantly reduced minute ventilation (36.1 [8.6] l/min to 28.1 [5.9] l/min), cardiac output (7.5 [2.3] l/min to 6.5 [1.9] l/min), and subjective scores for fatigue and breathlessness (13.9 [3.1] to 11.5 [3.5]) compared with room air intermediate changes were observed with 30% inspired oxygen. Increased inspired oxygen concentrations can improve exercise performance acutely and modify the ventilatory response to exercise in patients with heart failure. Hyperoxia reduces ventilatory response and circulatory demand while maintaining oxygen delivery at a given workload. The potential benefits of increased inspired oxygen concentrations in the treatment of chronic heart failure merit further assessment.


Incidence of cancers of the larynx and lung near incinerators of waste solvents and oils in Great Britain. The Small Area Health Statistics Unit (SAHSU) is a new independent facility for the investigation of disease near industrial installations in the UK. SAHSU analysed the incidence of cancers of the larynx and lung near the incinerator of waste solvents and oils at Charnock Richard, Coppull, Lancashire (which operated between 1972 and 1980) and nine other similar incinerators in Great Britain, after reports of a cluster of cases of cancer of the larynx near the Charnock Richard site. Postcoded cancer registration data were available for 1974-84 in England and Wales and 1975-87 in Scotland. Lag periods of 5 and 10 years were used between start-up (or first registration) of the incinerators and cancer incidence. Standardised observed/expected (O/E) ratios were assessed within 3 km and 3-10 km of each site and then aggregated over all sites. Expected values were based on national rates (regionally adjusted) with and without stratification by Carstairs' index, a measure of the socioeconomic profile of areas that uses census data for enumeration districts. Data were also assessed over a range of circles up to 10 km to test for trend in O/E ratios with distance. For Charnock Richard, none of the O/E ratios within 3 km or from 3-10 km differed significantly from unity, for either cancer or lag period. In the analysis of all sites with stratification by Carstairs' index, none of these O/E ratios differed significantly from unity for the two cancers. There was no evidence of decreasing risk with distance from the sites of either cancer. We conclude that the apparent cluster of cases of cancer of the larynx reported near Charnock Richard was unlikely to be due to its former incinerator.


Effects of passive immunization of growth hormone-releasing hormone and somatostatin on growth hormone secretion under conditions of high somatostatin tone. Pulsatile GH secretion decreases during food-deprivation in the rat. It has been hypothesized that this decrease is due to elevated hypothalamic somatostatin secretion. This is based on the observation that GH increases in food-deprived rats following removal of endogenous somatostatin using passive immunization techniques. Cognizant of the important stimulatory effects of growth hormone-releasing hormone (GHRH) on GH secretion, we sought to determine if this neuropeptide plays any role in mediating GH secretion in food-deprived rats. Male rats were prepared with indwelling venous catheters using sodium pentobarbital anesthesia seven days prior to experimentation. Animals were food-deprived for 72 h, after which control blood samples were drawn from -60 to 0 min. One group was then treated with normal rabbit serum (NRS), while a second group was treated with GHRH antiserum (GHRHab). At 55 min all animals received somatostatin antiserum (SSab). No animal exhibited any spontaneous GH peak during the one hour control period or in the subsequent one hour period following the administration of GHRHab or NRS. Absence of GH pulsatility during food-deprivation, coupled with no decrease in GH levels in food-deprived rats treated with GHRHab suggest that diminished GHRH pulsatility is likely during food-deprivation. Subsequent treatment of these animals with SSab resulted in an identical 2.5 fold increase in GH concentrations. This result suggests that GHRH is not involved in the GH rebound following somatostatin withdrawal in food-deprived rats.


Drugs affecting microtubule dynamics increase alpha-tubulin mRNA accumulation via transcription in Tetrahymena thermophila. In cultured mammalian cells, an increase in the amount of tubulin monomer due to treatment with a microtubule-depolymerizing agent results in a rapid decline in tubulin synthesis. This autoregulatory response is mediated through a posttranscriptional mechanism which decreases the stability of tubulin message with no change in transcriptional activity of tubulin genes. Conversely, treatment with a microtubule-polymerizing drug, such as taxol, results in a slight increase in the synthesis of tubulin. Surprisingly, we find that two microtubule-depolymerizing agents, colchicine and oryzalin, actually cause an increase in alpha-tubulin synthesis and alpha-tubulin message in starved Tetrahymena thermophila. This increase is paralleled by an increase in transcription of alpha-tubulin sequences measured by run-on transcription, while the half-life of tubulin message measured by decay in the presence of actinomycin D does not change appreciably. Treatment of starved cells with taxol also produces an increase in alpha-tubulin synthesis via an increase in message abundance due to an increase in transcription of the alpha-tubulin gene. These results indicate that tubulin synthesis in T. thermophila is regulated very differently than in cultured mammalian cells.


The effects on survival of early treatment of human immunodeficiency virus infection. Zidovudine has been shown to prolong survival in patients with the acquired immunodeficiency syndrome (AIDS) and, in persons with human immunodeficiency virus (HIV) infection but not AIDS, to delay the progression to AIDS. However, it is still uncertain whether treatment before the development of AIDS prolongs survival. We analyzed data from a cohort of 2162 high-risk men who were already seropositive for HIV type 1 (HIV-1) and 406 men who seroconverted from October 1986 through April 1991. There were 306 deaths. The probabilities of death were compared among men at similar stages of disease who began zidovudine therapy before the diagnosis of AIDS and among those who did not. Relative risks of death were calculated for each of five initial disease states on the basis of CD4+ cell counts and clinical symptoms and signs appearing over follow-up periods of 6, 12, 18, and 24 months. Adjustments were also made for the use of prophylaxis against Pneumocystis carinii pneumonia (PCP). After we controlled for CD4+ cell count and symptoms, the use of zidovudine with or without PCP prophylaxis before the development of AIDS significantly reduced mortality in all follow-up periods. The relative risks of death were 0.43 (95 percent confidence interval, 0.23 to 0.78) at 6 months, 0.54 (95 percent confidence interval, 0.38 to 0.78) at 12 months, 0.59 (95 percent confidence interval, 0.44 to 0.79) at 18 months, and 0.67 (95 percent confidence interval, 0.52 to 0.86) at 24 months. After we adjusted for the effects of PCP prophylaxis, zidovudine alone significantly reduced mortality at 6, 12, and 18 months (relative risks, 0.45, 0.59, and 0.70, respectively), but not at 24 months (relative risk, 0.81). Among zidovudine users, those who also used PCP prophylaxis before the development of AIDS had significantly lower mortality at 18 and 24 months than those who did not (relative risks, 0.62 and 0.60, respectively). The results of this study support the hypothesis that in HIV-1 infection, early treatment with zidovudine and PCP prophylaxis improves survival in addition to slowing the progression to AIDS.


Expression of the CD4 gene requires a Myb transcription factor. We have analyzed the control of developmental expression of the CD4 gene, which encodes an important recognition molecule and differentiation antigen on T cells. We have determined that the CD4 promoter alone functions at high levels in the CD4+ CD8- mature T cell but not at the early CD4+ CD8+ stage of T-cell development. In addition, the CD4 promoter functions only in T lymphocytes; thus, the stage and tissue specificity of the CD4 gene is mediated in part by its promoter. We have determined that a Myb transcription factor binds to the CD4 promoter and is critical for full promoter function. Thus, Myb plays an important role in the expression of T-cell-specific developmentally regulated genes.


The rationale for continuous dopaminergic stimulation in patients with Parkinson's disease. Continuous dopaminergic stimulation has been shown to stabilize motor fluctuations in patients with advanced Parkinson's disease (PD) who do not respond to more conventional forms of therapy. Levodopa infusions confer immediate benefit as a direct result of maintaining steady plasma levodopa concentrations. Fluctuations of synaptic dopamine inherent in the usual oral treatment of PD might result in deleterious postsynaptic changes. Some of these presumed receptor alterations might revert as a consequence of continuous levodopa infusion.


Initiating treatment of Parkinson's disease. Treatment of Parkinson's disease (PD) can be divided into two categories: symptomatic therapy (restoring dopamine levels toward normal and reversing functional disability) and preventive therapy (interfering with the pathophysiologic mechanism of PD to prevent or decrease the rate of progression of the disease). Regarding symptomatic treatment, although anticholinergic preparations generally are considered effective for the symptoms of tremor and rigidity without altering bradykinesia, their effectiveness is limited and adverse reactions are common; their role should be restricted to use as adjuvants to levodopa therapy. Amantadine has been shown to be as effective as anticholinergics, but it lacks long-term efficacy. Dopamine agonists--bromocriptine, pergolide mesylate and lisuride in Europe--are not as effective as levodopa and therefore rarely are used as initial therapy; their proposed role, too, is as adjuvants to levodopa therapy. Levodopa is the most effective drug presently available for the treatment of PD; its introduction is accompanied by rapid and dramatic reduction of symptoms and signs. Initial adverse reactions are not usually a major problem; and although there is speculation that initiation of therapy should be delayed because of possible long-term complications, clinically distinguishing these from problems related to disease progression itself is difficult. The possibility that nigral cell death is mediated by oxidative mechanisms provides the basis for considering antioxidant therapy as protective treatment; selegiline, an antioxidant, has been found to delay the need for symptomatic therapy. It is suggested that initial treatment of Parkinson's disease begin with both preventive therapy with selegiline and symptomatic treatment with the sustained-release preparation of levodopa, which may be associated with fewer long-term complications.


Fatal familial insomnia: a second kindred with mutation of prion protein gene at codon 178. Fatal familial insomnia (FFI), a condition characterized by inability to sleep, dysautonomia, motor disturbances, and selective thalamic atrophy is a prion disease linked to a GAC----AAC mutation at codon 178 of the prion gene. These data were obtained from one kindred. We now report a second kindred affected by FFI and carrying the same mutation. The finding of the same disease phenotype and genotype in a second family further validates FFI as a distinct disease entity and a phenotype of the GAC----AAC mutation at codon 178 of the prion gene.


Somatostatin analog in managing postgastrectomy duodenal stump leak. Somatostatin and its long-acting analog have a host of activities and potential applications in medicine today. Somatostatin is a naturally occurring peptide containing 14 amino acids. The following is one example of the many uses of somatostatin in the surgical patient.


The v-erbA oncogene requires cooperation with tyrosine kinases to arrest erythroid differentiation induced by ligand-activated endogenous c-erbA and retinoic acid receptor. The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), cooperates with tyrosine kinase oncogenes in erythroblast transformation. Here we show that the ligand-activated, endogenous retinoic acid receptor (RAR-alpha), in cooperation with c-erbA/TR-alpha, efficiently reverses the transforming effect of kinase oncogenes, overcoming oncogene-induced self-renewal by triggering terminal differentiation of the transformed cells into healthy erythrocytes. This differentiation induction was accompanied by up-regulation of erythrocyte gene expression. Similarly, RAR-alpha and over-expressed exogenous c-erbA/TR-alpha efficiently abolished the differentiation arrest caused by v-erbA, while the low levels of endogenous TR-alpha had no effect. In contrast, transformation by v-erbA plus a kinase oncogene was not affected at all by ligand-activated endogenous or over-expressed exogenous TR-alpha and RAR-alpha. These results suggest that oncogene cooperation is required to protect leukemic erythroblasts from differentiation induction via endogenous, nuclear hormone receptors. Endogenous c-erbA/TR-alpha and RAR-alpha apparently cooperated in abolishing erythroblast self-renewal and inducing differentiation, since the respective ligands acted in a synergistic fashion, and overexpressed, non-ligand-bound c-erbA/TR-alpha suppressed endogenous RAR-alpha function in differentiation induction. Genetic evidence is presented that this functional cooperation requires the receptor dimerization domain, suggesting that TR-alpha/RAR-alpha heterodimers play a role in regulation of erythroid differentiation.


Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action. The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.


Increased expression of genes from growth factor signaling pathways in glioblastoma cell lines. The concept of autocrine stimulation of cell proliferation postulates growth autonomy by acquisition of the ability to produce and respond to growth factors. Overproduction of several growth factors in a variety of human tumors and cell lines derived from these tumors has been reported. We have screened several cell lines derived from glioblastomas for anomalies in the expression of genes encoding transforming growth factor alpha (TGF-alpha), TGF-beta, basic fibroblast growth factor (bFGF) and its high-affinity receptor, flg. Compared with normal human brain tissue, we observed a generalized elevation in the levels of expression of these genes in glioblastoma cell lines and an SV40-transformed human astroglial cell line. Overexpression of these genes does not appear to be merely a reflection of the proliferative state of transformed cells since some other human tumor cell lines, when analysed for the expression of TGF-beta and bFGF, did not show a significant increase in these transcripts. The specificity of the elevated transcription of TGF-alpha, TGF-beta, bFGF and flg in glioblastoma cell lines is further suggested by the fact that the transcription of the proto-oncogene c-erbB2, which is overproduced in breast tumor cell lines, was not elevated in glioblastoma cell lines. Increased expression of growth factors, which are potent mitogens and angiogens, and/or their receptors may have critical roles in autonomous proliferation as well as neovascularization of glioblastomas.


Three distinct regions involved in 3p deletion in human lung cancer. The 3p deletion was first noted by cytogenetic analysis and was later confirmed by several independent studies using restriction fragment length polymorphism (RFLP) probes. As an initial step towards positional cloning (reverse genetics) of the tumor-suppressor gene(s) on 3p, a detailed analysis of the minimum deleted region(s) on 3p was performed with 13 RFLP probes and 48 paired human lung cancer samples. All nine small-cell lung cancer cases (100%) and 31 of 39 non-small-cell lung cancer cases (79%) showed allelic loss at one or more loci mapped on 3p. We show here that three distinct regions on 3p appear to be frequently deleted in lung cancer. These regions include 3p25, 3p21.3 and 3p14-cen. The present study should warrant future work focusing on these chromosomal regions on 3p, and may ultimately lead to the isolation of tumor-suppressor genes involved in the pathogenesis of lung cancer.


Surface expression of erbB-2 protein is post-transcriptionally regulated in mammary epithelial cells by epidermal growth factor and by the culture density. The control of expression of the erbB-2 protein was examined in two mammary epithelial cells lines, HC11 and 31E. The erbB-2 protein content varied dramatically depending upon cell density and upon the presence of epidermal growth factor (EGF) in the culture medium. The changes in protein content were not due to variation in the erbB-2 mRNA level. Analysis of the metabolic turnover of the erbB-2 protein showed that its rate of degradation was two- to threefold higher in cells growing at low density than in cells confluent for 2 days. The addition of EGF to the culture medium caused an increase in the phosphoamino acid content and an increase in the turnover of the erbB-2 protein. Cell fractionation experiments were performed, and a shift in the cellular localization of the erbB-2 protein towards the lysosomal compartment in EGF-treated HC11 cells was found. This is reflected by an increase in the degradation rate of the erbB-2 protein. These findings suggest that in mammary epithelial cells the stability of the erbB-2 protein is an important regulatory control point in determining the level of the protein. The degradation rate is sensitive to cell confluency and is controlled by EGF receptor activity.


Scc-1, a novel colon cancer susceptibility gene in the mouse: linkage to CD44 (Ly-24, Pgp-1) on chromosome 2. Mutations of proto-oncogenes and tumor-suppressor genes lead to neoplastic development. Some germline mutations of these genes increase the tumor susceptibility of their carriers, but the relationship between genes controlling tumor susceptibility and the known oncogenes and tumor-suppressor genes remains unelucidated. Moreover, as tumor susceptibility in mouse is controlled by multiple genes, their identification has been virtually impossible. We therefore developed a new system, the recombinant congenic strains (RCS), which separates individual susceptibility genes into different RC strains, thus facilitating their analysis. To map genes controlling the development of colon cancer, we used the Balb/c-c-STS (CcS/Dem) RC strains. Owing to several unidentified genes, Balb/cHeA mice are relatively resistant and STS/A mice highly susceptible to 1,2-dimethylhydrazine-(DMH)-induced colon adenocarcinomas. Each CcS/Dem strain carries a different subset of about 12.5% of genes of the STS strain on the Balb/c background, and individual STS susceptibility genes became segregated into different RC strains. Using CcS-19, one of the highly susceptible RC strains, we mapped a novel colon tumor susceptibility gene, Scc-1, different from the oncogenes and tumor-suppressor genes known to be involved in colon tumorigenesis, in the vicinity of CD44 (Ly-24, Pgp-1) on chromosome 2. The mapping of the Scc-1 gene indicates that the RCS system can be used to map and study the presently unknown genes which control cancer development.


The nuclear oncogenes v-erbA and v-ets cooperate in the induction of avian erythroleukemia. The nuclear oncogenes v-erbA and v-ets are known to cooperate with other viral oncogenes in the induction of avian erythroleukemia. Thus, in the case of avian erythroblastosis virus (AEV), v-erbA enhances the effect of the tyrosine kinase-encoding v-erbB oncogene by blocking the terminal differentiation of erythroid cells. In the case of E26 virus a fusion of the product from v-ets to that of the nuclear oncogene v-myb is a prerequisite for leukemogenicity. Here we show that an artificial virus carrying both v-erbA and v-ets induces a rapid, acute erythroleukemia phenotypically similar to that induced by AEV. In contrast, virus constructs containing either v-erbA or v-ets alone are non-leukemogenic, although they are capable of transforming erythroid cells in vitro. Analysis of in vitro-transformed cells showed that v-erbA induces a block of differentiation without abrogating dependence on anemic serum, while v-ets predominantly causes anemic serum independence. As expected, cells transformed by both oncogenes exhibit an increased proliferative potential, are blocked in differentiation and are anemic serum independent. These data demonstrate that two separately expressed nuclear oncoproteins can complement each other in vitro and in vivo. They also show that the v-Ets protein on its own can contribute to leukemogenesis.


[Alpha interferon treatment in hairy cell leukemia]. Since 1984 alpha-interferon (alpha IFN) has been the treatment of choice in cases of hairy-cell leukaemia. However, eight years' experience shows that, although a large majority of the patients are improved, they are not cured. Thus, a new type of cytostatic drug, perhaps capable of cure as well, may replace alpha-IFN for the treatment of hairy-cell leukaemia in the future.


[Interferon treatment in neuroendocrine tumors]. Neuro-endocrine tumours of the abdomen have long constituted a therapeutic challenge. Although characterised by slow growth rates, these tumours produce peptides and amines which in turn give rise to more or less severe clinical symptoms. Surgery has been deemed the treatment of choice, and is to be considered even in cases of metastasising disease. Medical treatment includes chemotherapy, and treatment with somatostatin analogues and interferons. By means of long-term treatment with alpha-interferon (leukocyte interferon), the disease can be controlled for long periods.


Pharmacologic treatment of attention deficit hyperactivity disorder. This article describes the role of psychostimulant medication in the treatment of attention deficit hyperactivity disorder. Included are the drugs' putative mechanisms of action, pharmacology, toxicology, indications for their use, short-term and long-term actions, adverse effects, specific dosing regimens, therapeutic monitoring techniques, alternative medications, and drug interactions.


Schizophrenia.

Schizophrenia. Schizophrenia occurring in childhood and adolescence has similar diagnostic, prognostic, and treatment ramifications as those noted with adult-onset schizophrenia. In assessing a child or adolescent suspected of having schizophrenia, care must be given to document DSM-III-R diagnostic criteria within the developmental framework of the patient's functioning, while thoroughly evaluating for other potentially confounding disorders or conditions. Antipsychotic therapy is the only specific treatment for schizophrenia, and should be a fundamental component with a multimodal treatment program that also addresses the psychological, social, and educational needs of the patient and his or her family. Strategies for medication management vary depending on several factors, including the stage of the disorder, noted or potential side effects, and the response of the patient to treatment, and need to be coordinated for the long term by a child or adolescent psychiatrist familiar with the diagnosis and treatment of schizophrenia in this age group.


The pharmacologic treatment of eating disorders. There is substantial evidence that antidepressant medication is significantly superior to placebo in the short-term treatment of bulimia nervosa. Further work is needed to determine long-term outcome of patients with bulimia nervosa who receive antidepressant treatment, the role of antidepressants in patients who are receiving psychological treatment, and the utility of sequential medication trials. In the treatment of anorexia nervosa, there is little evidence that psychotropic medications, including antipsychotics, antidepressants, and cyproheptadine, are of significant benefit to most patients who are in the acute phase of treatment and are receiving behavioral treatment to promote weight gain. There is preliminary evidence that antidepressant medication, specifically fluoxetine, may be useful in preventing relapse in weight-recovered patients with anorexia nervosa, but this has not yet been documented in randomized, double-blind, controlled trials.


The stimulants.

The stimulants. Stimulants are the most commonly prescribed psychotropic medications in child psychiatry, used generally for the treatment of attention deficit hyperactivity disorder (ADHD). In this article, the authors summarize the literature on the prevalence of use, neurobiology, and pharmacology of stimulants. Likewise, recent studies on the use of stimulants are reviewed, such as their use in specific ADHD populations including those with conduct disorder, girls, preschoolers, adolescents, and adults. Clinical guidelines for the management of children and adolescents receiving stimulants are offered, and treatment strategies are delineated for ADHD subjects with comorbidity and medication-induced adverse effects.