Effect of genetic dilution on development of diabetes, impaired glucose tolerance and in vitro glucose oxidation in LA/N-cp x SHR/N-cp F1 hybrid rats. 1. To determine the effects of gene dilution on development of IGT, NIDDM and in vitro glucose oxidation, heterozygous lean LA/N-cp female and SHR/N-cp male rats were mated, and F1 offspring studied at periodic intervals to determine the prevalence of obese and diabetic traits. 2. Obesity occurred in 25% of offspring by 5 weeks of age, consistent with inheritance of the autosomal recessive cp trait. 3. IGT occurred in all obese male F1, 67% of obese female F1, and 18% of the lean male F1 rats by 5 months of age, and diabetes occurred in 80% of male obese and 17% of female obese rats from 6 months of age. Glycosuria occurred with glucose intolerance, and was more severe in rats with NIDDM than IGT. 4. Rates of in vitro glucose oxidation were greater in diaphragm and adipose tissue, and were greater in the presence of insulin (100 mu Units/ml) in obese female but not obese male F1 rats. 5. These results indicate that the development of glucose intolerance is more prominent in male than in female F1 rats, that the progression of IGT to NIDDM occurs later in life in the F1 hybrid than in the SHR/N-cp strain from which the diabetic trait was transmitted, and that genetic dilution of the diabetic trait via hybrid breeding results in a delay in the expression of NIDDM which is chronologically more similar to that which occurs in man.


Serum beta-carotene before and after beta-carotene supplementation. A two-month double-blind, placebo-controlled supplementation study of oral beta-carotene (20 mg daily) was conducted. Two hundred and twenty two 30-69 year old men were randomized into either a beta-carotene or placebo group, and serum samples were obtained at baseline, follow-up (2 months), and up to 12 weeks post-supplementation. Serum beta-carotene increased on average 10-fold in the beta-carotene group, from 0.53 +/- 0.32 mumol/l (mean +/- SD) at baseline to 4.99 +/- 2.47 mumol/l at follow-up (P less than 0.0001), and beta-carotene levels remained elevated up to 12 weeks post-supplementation (0.61 +/- 0.15 mumol/l). No changes in serum retinol, alpha-tocopherol, or total cholesterol were observed. At baseline, serum beta-carotene levels were positively correlated with dietary beta-carotene (r = 0.29) and inversely correlated with body mass index and serum gamma-glutamyltransferase (r = -0.33 and r = -0.40, respectively). The inverse association with body mass index and serum gamma-glutamyltransferase persisted during active supplementation, whereas the positive association with dietary beta-carotene disappeared. In multivariate analysis, serum cholesterol was also positively associated with serum beta-carotene levels both before and after supplementation. Baseline serum beta-carotene was the factor most strongly associated (positively) with serum beta-carotene after supplementation. Our study highlights the importance of several factors which affect serum beta-carotene.


Vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in human penile corpus cavernosum tissue and circumflex veins: localization and in vitro effects. Localization and functional effects of vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM), two peptides derived from a common precursor molecule, were investigated in isolated preparations from human penile corpus cavernosum (CC) and circumflex vein (CV). VIP- and PHM-immunoreactivity (IR) was demonstrated in both CC and CV. The concentrations of VIP-IR and PHM-IR in CC tissue were 54.4 +/- 15.3, and 42.0 +/- 7.5 pmol g-1 wet weight respectively with a VIP/PHM ratio of 1.5 +/- 0.4 (mean +/- SEM). The corresponding values for CV tissues were 28.0 +/- 7.7 and 9.6 +/- 2.6 pmol g-1 wet weight with a VIP/PHM ratio of 3.1 +/- 0.4. CC and CV displayed VIP- and PHM-IR confined to nerve fibres in close relation to bundles of smooth muscle cells and blood vessels in both tissues. In vitro, VIP and PHM had no effects in unstimulated tissue preparations. Both peptides concentration-dependently (10(-9)-10(-6) M) relaxed CC and CV preparations precontracted with 3 x 10(-6) M noradrenaline. In CC the maximum relaxant effect of VIP and PHM was 22 +/- 11% and 9 +/- 9% and in CV the corresponding values were 82 +/- 8% and 93 +/- 3% respectively. The present study supports the hypothesis of VIP and PHM as neurotransmitters and/or neuromodulators in the nervous control of penile erection.


Somatostatin does not block the effect of vasoactive intestinal peptide on bile secretion in man. The effects of intravenously administered somatostatin and vasoactive intestinal peptide (VIP) on bile secretion were studied in 10 patients with complete biliary fistulas. The two peptides were administered both separately and simultaneously. During the infusion of vasoactive intestinal peptide, bile secretion increased by 85%, whereas during somatostatin infusion it decreased by 40%. When the peptides were administered together, the VIP-induced choleretic effect was not reduced by somatostatin. Vasoactive intestinal peptide infusion increased bicarbonate concentration and output, whereas somatostatin had the opposite effect. The output of chloride also increased following vasoactive intestinal peptide infusion but decreased following somatostatin infusion. The concentration of chloride was unaffected by somatostatin whereas it was decreased by vasoactive intestinal peptide. The output of bile acids was unaffected by vasoactive intestinal peptide and decreased by somatostatin infusion, whereas total lipid concentration increased during somatostatin infusion and decreased when vasoactive intestinal peptide was added. It is concluded that, in man, somatostatin acts on the bile acid-dependent canalicular bile secretion and also, to some extent, on the ductular secretion, whereas vasoactive intestinal peptide acts strictly at the ductular level. The effects of the two peptides on bile secretion are independent of each other.


The effects of light on cyclic nucleotide metabolism of isolated cone photoreceptors. A procedure has been developed for the isolation of cone photoreceptors from the retina of the lizard Anolis carolinensis in order to study the effects of dark- and light-adaptation on the cyclic nucleotide metabolism of these visual cells. Incubation of the retina in N2 medium with hyaluronidase and DNAase allows us to obtain intact photoreceptors with good purity (85-90%), yields (greater than 2 x 10(5) cells per retina), and more than 95% of them excluding Trypan blue. cAMP levels are 20-fold higher than cGMP levels in cells from dark-adapted animals and are decreased by 35% upon exposure to light, whereas cGMP levels show no apparent change. However, both cAMP- and cGMP-phosphodiesterases, as well as adenylate and guanylate cyclase, are activated several-fold by light, but the enzymes involved in cGMP metabolism have higher Vmaxs than the cAMP related enzymes. The apparent constant levels of cGMP found in cone photoreceptors may result from our inability to detect the very fast changes that occur in these cells when they are exposed to light.


Origin of mutation in sporadic cases of haemophilia-B. Of the 45 haemophilia-B patients registered at the haemophilia centre in Malmö, Sweden, 24 are the sole members of their families to be affected, and in 13 of these 24 cases, ascendant relatives are available for study. Detection of the gene defect showed the mutation to be de novo in the proband in 3 of these 13 cases, and inherited from a carrier mother in the remaining 10 cases. All 10 carrier mothers were shown to have de novo mutations, as the patients' grandfathers were phenotypically and/or haematologically normal, and the grandmothers were non-carriers. Seven restriction fragment length polymorphisms (RFLPs) of the factor IX gene were used to determine whether the de novo mutations of the 10 carrier mothers were of paternal or maternal origin. In 6/10 cases, the RFLP patterns were informative, and indicated the mutation to be of paternal origin.


Epidemiology of thrombocytopenia in HIV infection. Thrombocytopenia is a known complication of human immunodeficiency virus Type-1 (HIV-1) infection, and more data need to be collected on its frequency, severity, and clinical sequelae. We determined the frequency of thrombocytopenia and its relationship to other HIV infection characteristics from a review of records of 1004 HIV-infected patients attending two outpatient clinics in Washington, D.C. The self-reported sources of HIV-1 exposure were male homosexual activity (68%), bisexual activity (10%), heterosexual activity (6%), and intravenous drug use (15%). Fifty-nine percent of the individuals were white, 37% were black and 94% were male. Fifteen percent had AIDS. Thrombocytopenia occurred more frequently in subjects with AIDS (21.2%) than in HIV-infected individuals who did not fit clinical criteria for AIDS (9.2%) (p less than 0.001). Patients with few CD4-positive cells and an advanced stage of disease were more likely to have low platelet counts: 30% with an absolute CD4 cell count lower than 200/mm3 vs 8% with CD4 counts between 200 and 500 (p less than 0.00001), and 18.5% with Stage IV disease compared to 7.6% in Stage II (p less than 0.001) had platelet counts less than 150,000/mm3. Thrombocytopenia was more frequent in white males and older subjects. Although subjects infected by heterosexual exposure had a lower frequency of thrombocytopenia, intravenous drug users and homosexual men exhibited similar frequencies of thrombocytopenia. Of all subjects with platelet counts less than 50,000/mm3, 40% reported bleeding and 1 died of an intracranial hemorrhage. Thrombocytopenia occurs frequently in HIV-infected people, primarily in those with AIDS, low CD4 cell numbers, and advanced stages of diseases.


Mediator concentrations in bronchoalveolar lavage fluid of patients with mild asymptomatic bronchial asthma. Bronchoalveolar lavage (BAL) was performed on 11 atopic patients with mild asymptomatic bronchial asthma and 11 healthy nonasthmatic volunteers. All asthmatic subjects had evidence of bronchial hyperresponsiveness to inhaled carbachol. The concentrations of leukotriene (LT) C4, LTD4, LTE4, LTB4, prostaglandin D2 (PGD2), platelet-activating factor (PAF) and histamine in BAL fluid measured. The leukotrienes were measured by radioimmunoassay following reverse phase high performance liquid chromatography. PGD2 concentrations were determined by radio immunoassay after Amprep C2 extraction. PAF was quantitated by means of in vitro aggregation of rabbit platelets and histamine content was measured using a single isotope radio-enzymatic assay. There was an increase in the levels of PGD2 and a decrease in the concentration of LTB4 in asthmatic lavage samples. There were no significant differences in the lavage concentrations of LTC4/D4/E4 and histamine between the two groups of individuals. PAF was undetectable.


Effects of celiprolol, a cardioselective beta-blocker, on respiratory function in asthmatic patients. The aim of this study was to compare the pulmonary effects of a single dose of celiprolol 400 mg, versus bisoprolol 20 mg and the combination of celiprolol 400 mg plus propranolol 40 mg versus placebo plus propranolol 40 mg. We conducted a double-blind randomized cross-over study in 10 stable asthmatic patients (mean age +/- SD 31 +/- 7 yrs) with forced expiratory volume in one second (FEV1): 2.5 +/- 0.7 l. A three-day washout period preceded each treatment period. Measurements of respiratory function were done before treatment and after 90, 120 and 180 min. There was a significant increase of FEV1 (+12%) and forced vital capacity (FVC) (+8%) after celiprolol (p less than 0.05) and a decrease of FEV1 (-9%) after propranolol. Concerning the combination, celiprolol inhibits the bronchoconstrictor effects of propranolol. We conclude that celiprolol has bronchosparing properties in asthmatic patients, and even improves some of the ventilatory parameters.


Nonadrenergic, noncholinergic airway inhibitory nerves. Nonadrenergic, noncholinergic (NANC) nerves, which cause relaxation of airway smooth muscle, have been described in several species including man. Stimulation of efferent vagus nerves during cholinergic and adrenergic blockade induces a pronounced bronchodilation in the cat. In more recent studies in man, capsaicin inhalation or mechanical irritation of the larynx, under conditions of cholinergic and adrenergic blockade, have been shown to cause a transient bronchodilator response. There is some evidence that neuropeptides such as vasointestinal peptide (VIP) or peptide histidine methionine (PHM) may be the neurotransmitter of NANC nerves, but this is not conclusive. Nitric oxide may be another neurotransmitter. In mild asthma, the NANC bronchodilator response is similar to that observed in normal subjects; on the other hand, a reduction in VIP immunoreactivity has been reported in more severe patients. The contribution of NANC dilator nerves in pathophysiological situations is not known, but their effect may be modulated during allergic responses. Use of antagonists or inhibitors of putative neurotransmitters, and molecular biological techniques will be useful in defining both the physiological and pathophysiological roles of NANC inhibitory nerves in the airways.


Pleuropulmonary changes during treatment of Parkinson's disease with a long-acting ergot derivative, cabergoline. A patient with Parkinson's disease, initially treated with bromocriptine and subsequently with cabergoline, developed progressive pleuropulmonary abnormalities during the latter therapy. These lesions even worsened for some weeks after interruption of cabergoline, which may possibly be related to the prolonged action of this drug. Thus cabergoline may cause similar pleuropulmonary abnormalities to bromocriptine.


The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus. The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical A alpha and A beta loci. Four genes, one alpha and three beta, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes.


Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish. Homeo box-containing genes (Hox) are expressed in restricted regions of vertebrate embryos and may specify positional information. The organization and expression patterns of these genes are highly conserved among different species, suggesting that their regulation may also have been conserved. We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgene expression, and examined the activities of two mammalian Hox genes, mouse Hox-1.1 and human HOX-3.3. We found that these Hox promoters are activated in specific regions and tissues of developing zebrafish embryos and that this specificity depends upon the same regulatory elements within the promoters that specify the spatial expression of these genes in mice. Our results suggest that the promoter activities have been remarkably conserved from fish to mammals. To study the regulation of Hox expression in the developing nervous system, we analyzed the promoter activities in spt-1 mutants that have a mesodermal deficiency. Our results suggest that interactions, probably with the paraxial mesoderm, differentially regulate the activities of Hox promoters in the developing nervous system.


[Properties and activation by lysophospholipids and fatty acid of membrane-bound guanylate cyclase in lymphocytes and erythrocytes from mouse spleen]. To clarify the properties of membrane-bound guanylate cyclase of lymphocytes and a functional role of lysophospholipids, the enzymic properties of membrane-bound guanylate cyclase and the effects of lysophospholipids and a free fatty acid with Ca2+ on the cyclase in lymphocyte and erythrocyte membrane fractions prepared from mouse splenic whole cells were examined. The membrane-bound guanylate cyclase activities of lymphocyte and erythrocyte membrane fractions from splenic whole cells were activated markedly by several lysophospholipids and linoleate. Lysophospholipids were divided into three groups according to their effects on the cyclase. 1) The first group of lysophospholipids exhibited the concentration-activity curve which was similar to that of linoleate. 2) The second group showed the curves which were markedly different in the presence and absence of Ca2+. 3) The third group had no effects on the enzyme activity. The membrane-bound guanylate cyclase activity in the erythrocyte membrane fractions was about 2 times stronger than that in the lymphocyte fractions. On the other hand, the membrane-bound guanylate cyclase activity in the erythrocyte fractions from peripheral blood was not enhanced by these substances. The marked activation of the guanylate cyclase by lysophospholipids and linoleate is considered to have important significance in the regulation of cGMP-mediated signal transduction.


The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14. The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.


Linkage mapping of human chromosome 10 microsatellite polymorphisms. Ten microsatellite DNA polymorphisms located on human chromosome 10 were regionally mapped using subchromosomal somatic cell hybrids and linkage analysis. The resulting order of the markers from pter-qter was [D10S89, D10S111], D10S107, D10S109, [D10S91, D10S110, D10S108, D10S88, D10S168], and D10S169. Order of the markers within brackets was uncertain, although the order given was most likely. The microsatellites were distributed along the chromosome from the proximal p arm to near qter, with an unlinked gap between D10S168 and D10S169.


The multi-locus H-2Dw16 region has an organization distinct from the Dd region. Genomic DNA blot analyses using probes derived from the BALB/c 3' flanking region of the Ld gene (Ld 3' fl-C) and from near the BALB/c D3d gene (50.2A) indicate that the B10.GAA37 mouse strain has a multi-locus D (Dw16) region distinct from the five-gene organization observed in the Dd and Dq regions. To isolate the Dw16 region class I genes, a genomic B10. GAA37-lambda EMBL3 library was generated and screened with probes that preferentially hybridize to K and D region class I genes. Hybridization analyses of the isolated lambda clones with Ld derived oligonucleotide probes suggested that one of the lambda clones contained the Lw16 gene, whereas several other lambda clones contained the Dw16 gene. The sequence of the Dw16 gene is most similar to that of the Dp gene, particularly in the 3' half. Furthermore, the Lw16 gene is quite similar in the 5' half and virtually identical in the 3' half to the Ld gene, indicating that Lw16, but not Dw16, is a member of the Ld gene family. Collectively, these data suggest that, through a D region recombination event, the novel Dw16 region may have been assembled from primordial counterparts of the Dp and Ld genes.


Tumor induction by the LTR, v-src, LTR DNA in four B (MHC) congenic lines of chickens. We report that the cloned DNA harboring the long terminal repeat (LTR), v-src, LTR proviral structure is tumorigenic in chickens of the Prague congenic lines. The growth rate of these tumors is by far the highest in the recombinant CC.R1 line, the B haplotype of which is composed of the B-F/L4 and B-G12 subregions originating from different naturally occurring haplotypes. Some of the tumors induced by the LTR, v-src, LTR DNA are repeatedly transplantable in syngeneic chickens, maintain unaltered provirus, and express v-src mRNA. Differences in the response to challenge with Rous sarcoma virus (RSV) and LTR, v-src, LTR DNA on a given experimental model are compared and possible involvement of an interaction between B-F/L and B-G region genes is considered. Regression of the LTR, v-src, LTR DNA-induced tumors did not prevent the formation and growth of tumors induced subsequently by RSV.


Mapping of the genes encoding tum- transplantation antigens P91A, P35B, and P198. Tum- comprises a class of genes, mutation of which in P815 tumor cells has led to the acquisition of new cytotoxic T cell-recognized epitopes. The cells carrying the mutant alleles have impaired tumorigenicity compared with their progenitors due to in vivo induction of a cytotoxic T-cell response specific for tum- antigens. Two tum- genes, P91A and P35B, were found to be single copy loci mapping to chromosomes 11 and 15 respectively. A third, P198, was found to map to chromosome 7 and to be a member of a small gene family with other members on chromosomes 13, 14, and 15. Multiple P198-related sequences were found in other mammalian species suggesting the P198 related gene family is a general feature of mammalian genomes.


Alleles of the rat T-cell receptor beta chain gene complex. Inbred rat strains provide a rich source of genetic diversity in immunologically relevant genes. We have characterized the alleles of one of these genes, encoding the rat T-cell receptor C beta 1 chain, by Southern blots and nucleic acid sequencing. The Cb1 gene segments from DA and LEW rats display complex allotypic variation: both coding and noncoding regions contain multiple nucleotide substitutions. In addition, there is a polymorphic insertion of a rat repetitive LINE element 3' to the coding region. The Cb1 alleles are one part of larger Tcrb haplotypes, containing V beta, D beta, and J beta elements; complete Cb1 genomic nucleotide sequences, and a partial list of the strain distribution of the two alleles, are described in this report.


Ca2+ mobilization in nontransformed ciliary nonpigmented epithelial cells. To investigate the calcium second messenger system in nonpigmented epithelial (NPE) cells, we studied drug-dependent cytosolic free Ca2+ concentration ([Ca2+]i) transients in cultured nontransformed human and rabbit NPE cells with a fluorescent Ca2+ indicator, fura-2, and a digital video-imaging system. The main findings of this study were: (1) The basal [Ca2+]i was 141.9 +/- 1.2 nM (mean +/- standard error of the mean, n = 401) in humans and 157.0 +/- 1.4 nM (mean +/- SEM, n = 346) in rabbits. (2) Isoproterenol (10(-4) M) had little effect on [Ca2+]i mobilization in both species. Norepinephrine (10(-4) M) and epinephrine (10(-4) M) increased [Ca2+]i in 56% and 78% of rabbit NPE cells by 1.8- and 2.1-fold of basal [Ca2+]i, respectively, but induced little [Ca2+]i change in human NPE cells. Carbachol (10(-3) M) elicited significant [Ca2+]i increase (more than 3-4-fold of basal level) in about 60-70% of NPE cells in both species. Heterogeneity was seen in the cellular response to these agonists. (3) Norepinephrine-induced response was blocked by phentolamine (10(-5) M), and the effect of carbachol was blocked by atropine (10(-4) M). (4) Time course of norepinephrine-induced [Ca2+]i change was primarily monophasic. In contrast, [Ca2+]i transients induced by carbachol were mostly biphasic. (5) The duration of carbachol- or norepinephrine-induced responses were shortened by the chelation of extracellular Ca2+ without affecting other parameters of the reaction. This study confirms the presence of the calcium signaling system in cultured nontransformed human and rabbit NPE cells.


Neurotransmitters and neuropeptides stimulate inositol phosphates and intracellular calcium in cultured human nonpigmented ciliary epithelium. The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerol pathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inositol phosphate formation by vasopressin (approximately 3-fold), carbachol (approximately 2-fold) and histamine (approximately 5-fold) was observed only after cells had been confluent for at least six days. In contrast, a response to bombesin (approximately 3-fold) declined with extended time in confluent culture. Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showed a close correlation between the EC50s of the different agonists when elevation of inositol phosphates was compared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmacologic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order of potencies for selective agonists and antagonists. The data suggest that the muscarinic receptor on human NPE cells is the M3 subtype, whereas the vasopressin receptor, as defined by its linkage to the inositol phosphate/diacylglycerol pathway, is the V1 subtype.


Recommendations of the International Roundtable Workshop on Bovine Spongiform Encephalopathy. Recommendations of the working party were summarized as follows: Determine the status in all countries of their national cattle herds with respect to BSE. Attempt to develop a test to recognize BSE-infected animals before they become clinically ill. Establish procedures to prevent spread of BSE agent into the cattle populations, especially by eliminating feeds containing rendered ruminant proteins. Review the rendering processes, identify the sources and destinations of rendered products, and suggest appropriate changes if needed. Especially needed are standardized rendering procedures in regard to use of organic solvents, temperature, and duration of heat treatment. Review import and export regulations to reduce the risk of spreading BSE and to maximize opportunities for safe trading in cattle and cattle products. The scrapie-free certification program of the USDA was supported, and similar programs might be considered by other countries. If BSE/scrapie is diagnosed in a given country, determine baseline incidence of CJD in those countries and consider contributing to an international registry. The WHO should address the problems of BSE, formulate policy, participate in and coordinate research, and provide training opportunities for veterinary and human health care workers from eastern European countries and developing nations. Government and private agencies should consider increasing support for research on transmissibility and pathogenesis of CJD, BSE, CWD, scrapie, and transmissible mink encephalopathy. Prepare and publish a critical neuropathologic review of all spongiform encephalopathies, naturally and experimentally transmitted, defining the characteristics of each disease in the various species known to be susceptible. Consider producing guidelines for the biological and pharmaceutical industries with regard to sourcing, collecting, and processing bovine and ovine materials.(ABSTRACT TRUNCATED AT 250 WORDS)


The effects of temperature change and transient hypoxia on auditory nerve fiber response in the goldfish (Carassius auratus). Temperature change and hypoxia produce consistent, reversible effects on the response of single auditory nerve fibers in the goldfish. Cooling and hypoxia produce reductions of a cell's spontaneous activity, sensitivity, most excitatory or best frequency (BF) at a given signal level, and overall responsiveness to acoustic stimulation. Warming above ambient temperatures increases a cell's spontaneous activity, sensitivity, BF, and responsiveness. Adaptation, or the tendency for responsiveness to decline with time during a stimulus, increases during hypoxia and cooling, and decreases during warming. The effects of temperature change and hypoxia on a fiber's BF are similar to the effects of overall sound level. Since BF normally increases with sound level, the BF-shift with temperature change and hypoxia can be understood as a change in sensitivity or the overall effectiveness of a stimulus at a given sound level. The effects on neural response of temperature change and hypoxia are probably due in part to changes in the release and replenishment of neurotransmitter at the synapses between hair cells and auditory nerve fibers.


Methylprednisolone restores sensitivity to beta-adrenergic agonists in Basenji-Greyhound dogs. This study investigated the effect of chronic methylprednisolone treatment on the ability of albuterol and aminophylline to inhibit methacholine-induced airway constriction in Basenji-Greyhound (BG) dogs in vivo. Pulmonary responsiveness to methacholine was measured in five untreated BG dogs and in the same dogs pretreated with albuterol or aminophylline (which has been shown in this model to release endogenous catecholamines). Each dog was studied before, during, and after daily subcutaneous methylprednisolone for 6 wk. Changes in pulmonary resistance and dynamic compliance with methacholine aerosol challenge were measured. Neither baseline pulmonary function nor pulmonary responsiveness to aerosolized methacholine was significantly altered by albuterol, aminophylline, or chronic methylprednisolone administration alone. However, pretreatment with albuterol or aminophylline significantly attenuated airway responses to methacholine in BG dogs chronically receiving methylprednisolone. Because the reduced sensitivity to albuterol and aminophylline was restored by chronic methylprednisolone treatment, we conclude that at least part of the beneficial effects of corticosteroids on airways in BG dogs is through modulation of beta-adrenergic function.


The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.


Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers. Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element.


Role of sulfhydryl groups in Y2 neuropeptide Y receptor binding activity. Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.


Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells. Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.


Structure and organization of the human transglutaminase 1 gene. Membrane-associated transglutaminases (TGase1) have recently been found to be common in mammalian cells, but it is not clear whether these derive from the same or different genes. In order to determine the complexity of this system, we have isolated and characterized the human gene (TGM1). The gene of 14,133 base pairs was found to contain 15 exons spliced by 14 introns. Interestingly, the positions of these introns have been conserved in comparison with the genes of two other transglutaminase-like activities described in the literature, but the TGM1 gene is by far the smallest characterized to date because its introns are relatively smaller. On the other hand, the TGase1 enzyme is the largest known transglutaminase (about 90 kDa), apparently because its gene acquired tracts that encode additional sequences on its amino and carboxyl termini that confer its unique properties. Southern blot analyses of total human genomic DNA cut with several restriction enzymes reveal only one band. Use of human-rodent cell hybrid panels and chromosomal in situ hybridization with biotin-labeled probes revealed that the human TGM1 gene maps to chromosome position 14q11.2-13. Such data suggest there is a single gene copy per haploid human genome. Comparisons of sequence identities and homologies indicate that the transglutaminase family of genes arose by duplications and subsequent divergent evolution from a common ancestor but later became scattered in the human genome. Although our present Southern blot and chromosomal localization studies revealed no restriction fragment length polymorphisms, comparisons of published sequences and our genomic clone indicate there are two sequence variants for TGase1 within the human population. The rare smaller variant contains a two-nucleotide deletion near the 5'-end, uses an alternate initiation codon, and differs from the common larger variant only in the first 15 amino acids. Furthermore, the DNA sequences of intron 14 possess several tracts of dinucleotide repeats that by polymerase chain reaction analysis show wide size polymorphism within the human population. Accordingly, this gene system constitutes a useful polymorphic marker for genetic linkage analyses.


Visualization of purified fibronectin-transglutaminase complexes. It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.


Identification of a new squamous cell differentiation marker and its suppression by retinoids. Rabbit tracheobronchial epithelial cells (RbTE) can undergo squamous cell differentiation under defined culture conditions and, therefore, have been used as a model to study the regulation of squamous cell differentiation markers. In the present study, we identified a 20-kDa protein, designated rSQ20, in the serum-free growth medium conditioned by RbTE cells undergoing squamous cell differentiation. The protein was also found in extracts of squamous differentiated cells. rSQ20 was labeled by cells incubated with [35S]methionine but not with [3H]glucosamine, suggesting that it is not a glycoprotein. Undifferentiated cells did not produce this protein. rSQ20 was detected in the conditioned medium of RbTE cells after they reached a confluent and growth-arrested state, and thereafter its level increased markedly and concurrently with an increase in type I (epidermal) transglutaminase, an established marker of squamous cell differentiation. rSQ20 found in concentrated conditioned medium of squamous differentiated RbTE cells was eluted from a gel filtration column as a protein of 20 kDa, similar to that found by gel electrophoresis under denaturing conditions, suggesting that it is not a multimeric protein. A protein with an apparent molecular weight of 16 kDa (rSQ16), probably the product of partial proteolysis of rSQ20, was often found in various amounts in the conditioned medium of differentiated RbTE cells. beta-All-trans retinoic acid and other vitamin A analogues (retinoids), which suppress squamous cell differentiation, inhibited the expression of rSQ20 in RbTE cells. RbTE cells immortalized by transfection with SV40 large T antigen as well as malignantly transformed derivatives obtained from the immortalized cells by further transfection with v-Ha-ras secreted SQ20 and SQ16 when grown to high cell densities although their squamous differentiation was impaired. An analogous protein with an apparent molecular weight of 16 kDa, designated hSQ16, was detected in the medium of differentiated normal human bronchial epithelial (NHBE) cells and normal human epidermal keratinocytes (NHEK). No such protein could be detected in the medium in which undifferentiated NHBE or NHEK cells were grown. These results suggest that rSQ20 and hSQ16 are new markers of squamous cell differentiation.


Target-specific outgrowth from human mesencephalic tissue grafted to cortex or ventricle of immunosuppressed rats. Human fetal mesencephalic tissue was grafted to rats with unilateral lesions of the nigrostriatal pathway. The animals were immunosuppressed with cyclosporine A. Grafts were placed either into the lateral ventricle ipsilateral to the lesion or in the cingulate cortex above corpus callosum. The grafts and newly formed fibers were visualized by immunohistochemistry with antibodies against tyrosine hydroxylase (TH) and the human Thy-1 glycoprotein. TH-positive fibers covered the total volume of striatum when the graft was placed either in the ventricle or in the cortex. When the transplant was located in the ventricle, TH-positive cells migrated from the graft into host striatum. No cell migration was seen into any other areas than striatum. Cortex and septum were sparsely reinnervated by the graft, but not to a density higher than that normally seen. Globus pallidus was totally devoid of TH-positive fibers. When the graft was placed in cingulate cortex, fiber bundles penetrated through corpus callosum into either striatum, to arborize in its dorsal parts, or followed the medial side of the lateral ventricle to ventral limbic areas, where a fiber network also was formed. Human specific Thy-1-immunohistochemistry revealed positivity only on the lesioned side. These data suggest that dopamine neurons in human mesencephalic tissue, grafted to the rat brain, can migrate specifically into host striatum. Furthermore, TH-positive fiber outgrowth occurred only into dopamine denervated areas of the host, avoiding areas that are normally not innervated by nigral neurons, but also able to reach distant target cells.


Late asthmatic reaction decreased after pretreatment with salbutamol and formoterol, a new long-acting beta 2-agonist. The inhibitory effect of salbutamol and formoterol, a new long-acting beta 2-agonist for inhalation, on the late asthmatic reaction (LAR), was studied in 12 patients with allergic asthma. After a single-blind, placebo-treatment control, equipotent bronchodilating doses of inhaled salbutamol (500 micrograms) and formoterol (30 micrograms) were administered 30 minutes before bronchial allergen challenge in a double-blind randomized design. The early asthmatic reaction was completely inhibited by both drugs (p less than 0.01) but not by placebo. The LAR was also significantly inhibited by both drugs (p less than 0.01); formoterol was only slightly, but significantly, more effective than salbutamol (p = 0.04). In contrast to some earlier studies, the present study indicates an inhibitory effect of beta 2-agonists on the LAR.


The effect of formoterol on the late asthmatic phenomena in guinea pigs. We investigated the effects of formoterol, a new, long-acting, selective beta 2-adrenoceptor agonist, on the antigen-induced late asthmatic response (LAR) and airway inflammation in guinea pigs. Animals were sensitized by exposure to aerosolized ovalbumin (2% in saline). After antigen challenge, preceded by administration of an H1-receptor antagonist, specific airway conductance was measured with a two-chambered whole-body plethysmograph. An aerosolized solution of formoterol, isoproterenol, or saline was inhaled 15 minutes before challenge. Bronchoalveolar lavage (BAL) was performed 24 hours after challenge. The provocative concentrations of histamine required to decrease specific airway conductance by 50% were obtained before challenge, at 24 hours, and at 72 hours after challenge. The LAR (52.7% +/- 7.7% of the baseline; p less than 0.02) was observed 6 to 8 hours after antigen challenge. An increased cellular influx in BAL (mainly eosinophils and macrophages) and an increased bronchial responsiveness to histamine occurred 24 hours after antigen challenge. Formoterol completely inhibited the LAR and the cellular increase in BAL; however, isoproterenol failed to prevent either the cellular infiltration or the LAR. Formoterol also decreased the antigen-induced increase in bronchial reactivity. These findings suggest that formoterol has inhibitory effects on the underlying inflammatory processes in antigen-induced asthma in addition to prolonged bronchodilation.


Studies of bone marrow progenitor cells in lupus-prone mice. I. NZB marrow cells demonstrate increased growth in Whitlock-Witte culture and increased splenic colony-forming unit activity in the Thy-1-, lineage- population. Previous studies have demonstrated that NZB marrow can transfer features of autoimmunity. Therefore, we undertook a study of NZB marrow to determine whether it demonstrated any phenotypic abnormalities. In Whitlock-Witte cultures, NZB marrow cells generated nonadherent cells at low seeding densities, densities at which marrow from other strains did not generate nonadherent cells. In contrast, NZB marrow grew less well than controls in Dexter cultures. Inasmuch as the latter favor growth of granulocyte-macrophage precursors and the former B cells, these results suggest a possible skewing of NZB marrow cells toward lymphocyte production. Unfractionated marrow cells from NZB mice were found to produce 10-fold more splenic colonies in lethally irradiated recipients than marrow cells from control mice. This result was independent of the genotype of the recipient. When the progenitor Thy-1lo, Lin- marrow subpopulation was studied, NZB mice did not differ substantially from controls regarding splenic CFU. Therefore, Thy-1-, Lin- marrow cells were studied as a possible source of the excess splenic CFU in NZB mice. Indeed, the NZB Thy-1-, Lin- population contained 30-fold more splenic CFU than did the Thy-1-, Lin- population from control mice. These results suggest that NZB mice have unusual marrow progenitor cells; such cells may play a role in their autoimmune disease.


Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes. Monocytes play a critical role in defending the host against foreign organisms and in regulating the behavior of other cells. Monocytes circulate as nonadherent cells in the blood and migrate as adherent cells through tissues. Adhesion molecules mediate not only cell adhesion, but also migration, phagocytosis, and many other adhesion-dependent functions. Monocyte chemoattractant protein-1 (MCP-1) is thought to be responsible for monocyte recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation. In this study, immunofluorescence flow cytometry was used to determine whether MCP-1 can regulate the cell surface expression of adhesion molecules, particularly beta-2 and alpha-4 integrins and the leukocyte adhesion molecule-1. We found that MCP-1 induced expression of CD11c (p150,95 alpha-subunit) and CD11b (Mac-1 alpha-subunit), and caused little or no change of CD11a (lymphocyte function-associated Ag-1 alpha-subunit), very late activation Ag-4, or leukocyte adhesion molecule-1. We demonstrated that antibodies to beta-2 and alpha-4 integrins inhibited MCP-1-induced monocyte chemotaxis. We also showed that MCP-1 is capable of inducing IL-1 and IL-6, but not TNF production of monocytes. These results indicate that MCP-1 is not only a chemoattractant but also a novel cytokine with the capacity to regulate several parameters of monocyte function.


Distinct T cell specificities are induced with the authentic versus recombinant Plasmodium berghei circumsporozoite protein. Plasmodium berghei sporozoite (SPZ)-immune lymph node (LN) cells obtained from mice of different H-2 haplotypes were analyzed for the presence of circumsporozoite (CS) protein-reactive T cells in proliferative assays. Although lymphocytes from each strain responded in vitro to the priming Ag and to the soluble rCS protein, they did not respond to CS protein synthetic peptides. Parallel analysis of rCS protein-primed LN cells revealed that the two Ag are unequal in generating T cell specificities: although SPZ priming did not induce CS protein peptide-reactive T cells, priming with rCS protein did. Not being privy to the processing and presentation of SPZ Ag, we postulated that a different order of processing of the authentic, i.e., SPZ-associated CS protein vs soluble rCS protein might be responsible for the generation of different T cell specificities. Accordingly, authentic CS protein might not be processed by APC, or the processed fragments might obscure the recognition of smaller peptide fragments. Therefore, we subjected the SPZ to three cycles of a freeze/thaw procedure and used the denatured SPZ preparation for priming. We observed that contrary to priming with the authentic SPZ, denatured SPZ generated T cells reactive to some of the CS protein synthetic peptides. The hypothesis that each form of the SPZ Ag is subject to a unique Ag processing was also confirmed in experiments demonstrating a lack of recognition of the authentic CS protein by rCS protein-primed LN cells. Hence, the evidence presented in this work that complex protozoan Ag, such as Plasmodia, might present different requirements for Ag-specific T cell induction/activation not only enhances the basic understanding of the immune system, but is essential for the development of antimalaria vaccine(s). In addition, these observations support the hypothesis that the molecular context of the priming Ag influences the outcome of T cell specificities, by providing evidence that the authentic CS protein induces a T cell repertoire that is distinct from that induced by the rCS protein.


CD28-stimulated IL-2 gene expression in Jurkat T cells occurs in part transcriptionally and is cyclosporine-A sensitive. CD28 is a glycoprotein expressed as a homodimer on the surface of a major subset of human T cells. Previous studies have shown that proliferation of peripheral blood T cells involving the CD28 pathway is associated with cyclosporine A (CsA) resistant IL-2 gene expression. This pathway was shown to specifically regulate the stability of mRNA for several lymphokines including IL-2. We have investigated the expression of the IL-2 gene in the Jurkat cell line, J32 clone, induced by CD28 stimulation. Cross-linked anti-CD28 mAb alone was sufficient to induce the release of small amounts of IL-2 (256 U/ml). The CD28-mediated IL-2 release was enhanced with simultaneous engagement of CD28 and CD2 or CD28 and CD3 molecules. Hybrid constructs in which the human IL-2 gene 5' flanking region drives luciferase expression (pIL-2-Luc) were used to help delineate whether the CD28 pathway activates the IL-2 gene transcriptionally. Costimulation of cells with CD28 mAb and either PHA or CD2 mAb induced a 20- to 90-fold increase in the expression of pIL-2-Luc as well as IL-2 release. Costimulation with CD28 mAb plus PMA gave only five- to sevenfold increase in enhancer activity. In contrast, no enhancer activity was detected after stimulation with CD28 or CD2 mAb alone. Both IL-2 release and pIL-2-Luc activity were inhibited by CsA in J32 cells. The degree of CsA inhibition was concentration dependent and was similar in cells stimulated with either CD28 mAb or CD3 mAb. Maximum inhibition was achieved with 1 microgram/ml of CsA. Studies with internal deletion mutations of the IL-2 gene 5' flanking sequence revealed that as with stimulation through the TCR pathway, the CD28 pathway requires 5' flanking sequences located within 500 bp of the transcription start site. These results are the first direct evidence that the triggering of CD28 molecule is sufficient to induce IL-2 release in J32 cells. Furthermore these studies strongly indicate that IL-2 gene expression induced by CD28 stimulation occurs, in part, transcriptionally and is CsA sensitive in these cells.


Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice. The systemic administration of human rIL-6 to mice resulted in the regression of established, 3-day pulmonary micrometastases from two weakly immunogenic tumors, but not from a nonimmunogenic tumor, in the absence of observable toxicity. Although IL-6 alone failed to have a significant therapeutic impact on advanced, 10-day pulmonary macrometastases from weakly immunogenic tumors, substantial cure rates of mice could be achieved when this cytokine was combined with cyclophosphamide. Histologic analysis of the lungs of mice receiving IL-6 revealed infiltration with lymphoid cells during the regression of pulmonary nodules from a weakly immunogenic tumor. IL-6-mediated tumor regression could be abrogated after selective in vivo depletion of either CD4 or CD8 T cell subsets by the systemic administration of specific mAb. In vivo generation of tumor-specific CTL, but not of lymphokine-activated killer cells, was detected in the lungs of IL-6-treated mice during regression of pulmonary metastases. Collectively, these findings demonstrate a role for IL-6 in the treatment of established solid tumors that have the capacity to elicit T cell responses in the host. Differences in host cellular mechanisms involved in tumor regression mediated by immunotherapy using IL-6 vs IL-2 are discussed.


Excitatory transmitter amino acid release from the ischemic rat cerebral cortex: effects of adenosine receptor agonists and antagonists. The effects of selective adenosine receptor agonists [N6-cyclopentyladenosine (CPA) and N-ethylcarboxamidoadenosine (NECA)] and antagonists [8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazoline-5-im ine (CGS-15943A)] on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four-vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug-treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10(-10) M) and NECA (10(-9) M) significantly inhibited the ischemia-evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia-evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10(-6) M) or NECA (10(-5) M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia-evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS-15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia-evoked release of excitatory neurotransmitter amino acids via high-affinity A1 receptors, whereas coactivation of lower-affinity A2 receptors may block (or reverse) the A1-mediated response.


Dopaminergic modulation of glutamate release in striatum as measured by microdialysis. Glutamate and aspartate are the primary neurotransmitters of projections from motor and premotor cortices to the striatum. Release of glutamate may be modulated by dopamine receptors located on corticostriatal terminals. The present study used microdialysis to investigate the dopaminergic modulation of in vivo striatal glutamate and aspartate release in the striatum of awake-behaving rats. Local perfusion with a depolarizing concentration of K+ through a dialysis probe into the rat striatum produced a significant increase in the release of glutamate, aspartate, and taurine. The D2 agonist LY171555 blocked the K(+)-induced release of glutamate and aspartate, but not taurine, in a concentration-dependent manner. The D1 agonist SKF 38393 did not alter K(+)-induced release of glutamate and taurine, but did significantly decrease aspartate release. Neither agonist had any effect on basal amino acid release. The D2 antagonist (-)-sulpiride reversed the inhibitory effects of LY 171555 on K(+)-induced glutamate release. These results provide in vivo evidence for a functional interaction between dopamine, the D2 receptor, and striatal glutamate release.


N-methyl-D-aspartate antagonists block fos-like protein expression induced via multiple signaling pathways in cultured cortical neurons. c-fos mRNA and Fos-like protein(s) (FLP) are induced in cultured cortical neurons by glutamate, high K+, phorbol ester, basic fibroblast growth factor, Zn2+, and vasoactive intestinal peptide. Glutamate induction of c-fos mRNA and FLP is blocked by noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, and competitive NMDA antagonists, 4-(3-phosphonopropyl)piperazin-2-carboxylic acid and 2-amino-7-phosphonoheptanoate. These antagonists partially block high K(+)-, phorbol ester-, Zn(2+)-, and VIP-induced c-fos mRNA expression, but have no effect on bFGF-induced c-fos mRNA expression. However, both competitive and noncompetitive NMDA antagonists completely block FLP induction by all of these agents without affecting total protein synthesis. Therefore, these NMDA antagonists block FLP translation, without blocking c-fos transcription. It is hypothesized that NMDA receptor activation is required for translation of c-fos mRNA in cortical neurons after stimulation of multiple intracellular signaling pathways. It is possible that NMDA antagonists prevent cortical plasticity by blocking induction of the Fos protein that would normally be induced by neurotrophic factors, neurotransmitters, and neuromodulators.


Glutamate metabolism in rat cortical astrocyte cultures. Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.


Astrocyte heterogeneity: endogenous amino acid levels and release evoked by non-N-methyl-D-aspartate receptor agonists and by potassium-induced swelling in type-1 and type-2 astrocytes. The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.


1-Aminocyclopentane-trans-1,3-dicarboxylic acid induces glutamine synthetase activity in cultured astrocytes. In this study we have investigated the effect of excitatory amino acids on the activity of glutamine synthetase, a glial-specific enzyme that plays a key role in the regulation of glutamate concentration in the CNS. We found that of L-glutamate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, kainate, and 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), only the metabotropic glutamate receptor agonist trans-ACPD had an effect on glutamine synthetase specific activity in cultures of rat type I cortical astrocytes. Exposure of astrocytes to 1.0 mM trans-ACPD for 24 h resulted in an increase in glutamine synthetase activity to 149 +/- 11% of that in control cultures. This effect was concentration dependent, stereoselective, and blocked by cycloheximide. In addition, the increase in glutamine synthetase activity occurred at lower concentrations of trans-ACPD that did not produce morphological alterations or lysis of the astrocytes as measured by the lactate dehydrogenase content. These findings are consistent with the hypothesis that activation of the metabotropic excitatory amino acid receptor in astrocytes is coupled to the regulation of an enzyme essential to the metabolism and recycling of the excitatory transmitter L-glutamate.


Detection of migrated allogeneic oligodendrocytes throughout the central nervous system of the galactocerebrosidase-deficient twitcher mouse. Galactocerebrosidase-deficient oligodendrocytes of 'twitcher' (twi/twi) mice degenerate prematurely. Transplantation of normal bone marrow cells has been shown to alleviate symptoms and to prolong survival time. However, characteristic ataxia ('twitching') is not cured. In an attempt to improve further the condition of twitcher mice, allogeneic foetal liver cells were transplanted as a source of normal haemopoietic stem cells and supplemented with intracerebral transplantation of foetal brain cells. A reliable method was developed to detect donor-type cells in brain tissue. Bacteriophage lambda transgenic foetal mice were used as donors of both foetal liver and brain cells. Integrated copies of lambda DNA in donor cells were detected by in situ hybridization with biotinylated probes, which were then stained using streptavidin alkaline phosphatase. This technique was combined with immunohistochemistry to distinguish donor-type oligodendrocytes from macrophages. Immunoperoxidase staining with an antiserum to carbonic anhydrase-II produced dark perikarya of oligodendrocytes. The results demonstrated that local foetal brain cell grafts resulted in a wide dissemination of donor-type oligodendrocytes throughout the twitcher brain. The addition of a foetal brain cell graft to haemopoietic cell transplantation resulted in significantly prolonged survival of twitcher mice.


Expression of LFA-1/ICAM-1 in CNS lymphomas: possible mechanism for lymphoma homing into the brain. We examined a possible role for the adhesion molecules LFA-1 and ICAM-1 in localizing central nervous system non-Hodgkin's lymphomas (CNS-NHLs) to the brain. Fresh frozen sections from 12 monoclonal CNS NHLs (11 primary, one secondary) were stained with monoclonal antibodies to LFA-1 alpha chain (CD11a), beta chain (CD18) and, ICAM-1 (CD54). Additional staining made use of rat monoclonal antibodies to the human and mouse high endothelial venule antigens HECA 452 and MECA 79 and mouse ICAM-1. The expression of these same molecules was also studied in mice with severe combined immunodeficiency (SCID) mice, bearing intracranial human lymphoblastoid cells. Eleven of the CNS-NHL tumors expressed LFA-1 alpha (one strongly, one intermediate, nine weakly). Nine of the tumors weakly expressed LFA-1 beta.. Nine of twelve tumors weakly expressed ICAM-1. In six of seven tumors definite blood vessels stained for ICAM-1. Non-tumor brain from two patients and non-tumor cerebral blood vessels showed no staining with CD11a, CD18 or CD54 antibodies. Strong expression of LFA-alpha and LFA-beta as well as ICAM-1 was noted in human lymphoblastoid cells (LCLs)/SCID mouse CNS lymphomas. Tumor blood vessels in these mice stained for mouse ICAM-1. Normal SCID mouse brains showed no staining with CD11a, CD18, CD54 or mouse ICAM-1 antibodies. Human, human/mouse CNS lymphomas, normal human, and mouse brains showed no staining with either HECA 452 or MECA 79.(ABSTRACT TRUNCATED AT 250 WORDS)


Subcutaneous tocolytic infusion therapy for patients at very high risk for preterm birth. Patients with multiple gestations or recalcitrant preterm labor are at very high risk for preterm birth in spite of adequate tocolysis. Subcutaneous infusion of tocolytic medications on an ambulatory basis has been used in several small series and has effectively prolonged gestation. This retrospective analysis presents data from 992 patients at very high risk for preterm delivery who were prescribed this therapy. The amount of tocolytic medication was individualized by utilizing the patient's volume of distribution and clearance. Pharmacists adjusted the dosage based on uterine activity strips received by nursing personnel. The average basal rate was .073 +/- .020 mg/h. Patients received an average of seven scheduled boluses per day and 1.54 +/- .93 unscheduled boluses per week (.25 +/- .03 mg each). The therapy extended the gestation a mean of 38 +/- 23 days and average gestational age at delivery was 36.3 +/- 2.6 weeks with a mean birthweight of 2759 +/- 681 g. This study, utilizing a large number of patients, confirms earlier reports that for women at very high risk for preterm delivery subcutaneous tocolytic infusion therapy is beneficial. Prospective studies evaluating such treatment on a randomized basis are indicated.


A comparison of proliferating cell nuclear antigen (PCNA) immunostaining, nucleolar organizer region (AgNOR) staining, and histological grading in gastrointestinal stromal tumours. Gastrointestinal stromal tumours are lesions in which it is difficult to predict clinical outcome from the histological appearances. Sixty cases were studied using three different methods of assessing aspects of cellular proliferation; these were (i) immunostaining for proliferating cell nuclear antigen (PCNA), (ii) interphase nucleolar organizer region staining (AgNORs), and (iii) a histological grading system based on mitotic counts. Both PCNA immunostaining and AgNOR counts were found to correlate well with histological grading and all three methods independently showed good correlations with survival. This suggests that these proliferation-associated markers may be used as additional features to support histological grading in this relatively uncommon group of tumours.


Determination of the optimal ratio of linoleic acid to alpha-linolenic acid in infant formulas. The fatty acid composition of erythrocyte total lipids taken from a group of term infants 10 weeks after being fed a commercial infant formula with a high ratio of linoleic acid (18:2n-6) (LA) to alpha-linolenic acid (18:3n-3) (ALA) (19:1; LA, 14%; ALA, 0.7%; group A, n = 10) was compared with the fatty acid composition of erythrocytes from infants fed formulas that contained LA/ALA ratios reduced by either increasing ALA (4:1; LA, 13%; ALA, 3.3%; group B, n = 11) or decreasing LA (3:1; LA, 3.5%; ALA, 1.1%; group C, n = 8). Results were compared with those in an age-controlled group (n = 9) of breast-fed infants. Decreasing the LA/ALA ratio increased n-3 C20 and C22 fatty acid incorporation (formula B = 8.98% +/- 0.65%; formula C = 9.30% +/- 0.95%) relative to formula A (5.97% +/- 0.76%; p less than 0.05). Although docosahexaenoic acid (22:6n-3) (DHA) incorporation was highest in infants fed formulas B and C (4.78% +/- 0.45% and 4.48% +/- 0.49%, respectively) relative to formula A (3.47% +/- 0.46%; p less than 0.05), it did not reach levels found in breast-fed infants (6.55% +/- 1.23%; p less than 0.05). In addition, levels of arachidonic acid (20:4n-6) (AA) were lower in all formula-fed groups (p less than 0.05) relative to those in breast-fed infants. Based on some equations, it is predicted that AA levels in tissues of infants fed lower LA/ALA ratios would be reduced even further. Because both AA and DHA are probably essential for normal neural development of the infant, formulas with LA/ALA ratios below 4:1 are likely to result in fatty acid profiles notably different from those of breast-fed infants.


Immune serum markers and CD4 cell counts in HIV-infected intravenous drug users. We examined the association of three serum immune markers with CD4 cell counts in a large cohort of i.v. drug users with and without human immunodeficiency virus (HIV) infection. Levels of beta 2-microglobulin and neopterin were significantly elevated in HIV-infected subjects and increased in association with decline in CD4 cell counts (all p less than 0.001). Serum IgA levels in HIV-seropositive individuals were significantly elevated only when the CD4 cell count was less than 200/microliters (p less than 0.001). After controlling for HIV status and CD4 count, recent history of hepatitis was associated with significantly higher beta 2-microglobulin (p = 0.028) and marginally higher neopterin (p = 0.052) levels. There was no association of race, gender, or drug use patterns with levels of serum immune markers after controlling for HIV status and CD4 count. These data indicate that immune activation is coupled with immunosuppression in HIV-infected i.v. drug users. In addition, beta 2-microglobulin and neopterin levels are elevated in persons with a recent history of hepatitis but not in those with recent non-AIDS-defining bacterial infections. Markers of immune activation do not vary by race, gender, or drug use patterns among i.v. drug users.


Sequestration of organic cations by acidified hepatic endocytic vesicles and implications for biliary excretion. A number of cationic amine drugs that are taken up by liver and excreted into bile may accumulate in acidified intracellular organelles such as lysosomes and endosomes. These studies were undertaken to assess directly the uptake and accumulation of three types of model organic cationic amines by endocytic vesicles, and the role of vesicle acidification in this process. Uptake of tubocurarine (TC), vecuronium and tributylmethylammonium (TBuMA) by purified rat liver multivesicular bodies (MVB) (prelysosomal endocytic vesicles) was dependent upon MgATP, time and drug concentration. After 60 min, 52 to 81% of MVB cation content was dependent upon vesicle acidification (due to an electrogenic proton pump), but not upon an interior positive vesicle membrane potential. Nineteen to 42% of MVB cation content appeared due to binding to MVB membranes or to internal lipoproteins. Vesicle-to-medium ATP-dependent apparent concentration ratios for these three cations were 3.3 to 51. MVB uptake of these cations resembled uptake of methylamine, a tertiary amine known to distribute across organellar membranes according to pH gradients. By contrast, MVB uptake of the lipophilic quaternary amine methyldeptropine was not dependent upon MgATP or on development of MVB pH or membrane potential gradients. In further studies, TC, vecuronium and TBuMA were rapidly taken up by the isolated perfused rat liver and excreted in bile. Exposure to 250 mciroM primaquin (which partially alkalinized acidic endosomes and lysosomes) reduced accumulation of [3H]vecuronium in a lysosomal fraction by 23%, decreased perfusate disappearance of TC and TBuMA, but not of vecuronium, and decreased biliary appearance of all three cations. These studies suggest that acidified intracellular organelles sequester certain organic cationic drugs, possibly via a drug/proton antiporter, and/or diffusion followed by intravesicular protonation and trapping of tertiary amines. However, attempts at partial displacement of these drugs, accomplished through partial vesicle alkalization by primaquin, decreased excretion of TC, vecuronium and TBuMA, perhaps reflecting the small functional size of the displaceable organellar drug compartment and/or competition between primaquin and the organic cations for membrane transport processes.


Naloxone and norbinaltorphimine administered intracerebroventricularly antagonize spinal morphine-induced antinociception in mice through the antianalgesic action of spinal dynorphin A (1-17). Previously, a number of analgesic agonists, when administered i.c.v. to mice, were shown putatively to activate the release of dynorphin A (1-17) (Dyn A) in the spinal cord. Whether released endogenously or administered i.t., Dyn A produces an antianalgesic action against i.t. administered morphine. In the present study, the opioid antagonists, naloxone and norbinaltorphimine (N-BNI), were shown to activate the Dyn A system. Intracerebro-ventricular administration of both naloxone and N-BNI antagonized the antinociceptive effect of i.t. morphine in the mouse tail-flick test, an effect designated as an antianalgesic action. This antianalgesic action was demonstrated to be mediated by spinal Dyn A in the following ways: 1) the antagonistic effect of i.c.v. naloxone and N-BNI was eliminated by administration of small doses of i.t. naloxone and N-BNI, a unique situation where administration of the opioid antagonists at a second (i.t.) site reversed the antagonistic effect of opioid antagonists administered at the other (i.c.v.) site; 2) i.t. pretreatment with dynorphin antiserum prevented the antianalgesic effect; 3) morphine pretreatment (s.c., 10 mg/kg), which produces desensitization to the effect of spinal Dyn A, eliminated the antianalgesic effect; and 4) pretreatment with i.c.v. naloxone (3 hours) and N-BNI (24 hours) which presumably releases Dyn A produced desensitization to the antagonistic effect of i.c.v. naloxone and N-BNI as well as to the antianalgesic action of i.t. Dyn A. Taken together, the results indicate that both i.c.v. naloxone and N-BNI produced indirect antagonistic actions which were mediated at the spinal cord by the antianalgesic action of Dyn A.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of cyclosporine on colchicine secretion by the kidney multidrug transporter studied in vivo. The multidrug resistance (MDR) transport protein is a normal constituent of proximal renal tubules although its function has not been defined in vivo. We find that colchicine, an MDR substrate, is secreted into urine by a process which is distinct from the organic cation transporter responsible for tetraethylammonium and N-methylnicotinamide secretion. Cyclosporine (CsA), which reverses MDR in vitro presumably by inhibiting the MDR transporter, inhibits colchicine renal secretion but does not inhibit the net secretion of the organic cation, ranitidine, or the organic anion, p-aminohippurate. After CsA (2 mg/kg i.v.), colchicine renal clearance decreased from 6.23 +/- 0.46 to 3.58 +/- 0.31 ml/min.kg (P less than .05), glomerular filtration rate was unchanged (4.21 +/- 0.08 and 3.88 +/- 0.17 ml/min.kg, before and after CsA, respectively; P = .09) and colchicine secretory ratio decreased from 1.48 +/- 0.11 to 0.92 +/- 0.07 (P less than .05). Cremophor (CsA vehicle) increased colchicine renal clearance (6.77 +/- 0.29 to 7.7 +/- 0.3 ml/min.kg, P less than .05) and colchicine secretory ratio (1.425 +/- 0.071 to 1.621 +/- 0.061, P less than .05). The inhibition of colchicine secretion was long-lived lasting at least 30 hr after CsA. Thus, colchicine is actively secreted into urine by the multidrug transporter in vivo. CsA profoundly inhibits colchicine secretion into urine while having no effect on the secretion of the organic cation ranitidine or the organic anion p-aminohippurate.


A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices. Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)


Potent and systemically active aminopeptidase N inhibitors designed from active-site investigation. Derivatives of amino acids bearing various zinc-coordinating moieties (SH, COOH, CONHOH, and PO3H2) were synthesized and tested for their ability to inhibit aminopeptidase N (APN). Among them, beta-amino thiols were found to be the most efficient with IC50's in the 11-50 nM range. These results suggest that the S1 subsite of APN is a deep but not very large hydrophobic pocket, optimally fitting side chains of moderate bulk endowed with some degree of freedom. The iv administration of the inhibitors, alone, did not induce antinociceptive responses on the hot plate test in mice. However, in presence of 10 mg/kg acetorphan, a prodrug of the neutral endopeptidase inhibitor thiorphan, these compounds gave a large increase in the jump latency time with ED50's of 2 and 2.4 mg/kg for the disulfides of methioninethiol H2NCH(CH2CH2SCH3)CH2S]2 and S-oxomethioninethiol [H2NCH(CH2CH2S(O)CH3)CH2S]2, respectively. These results show that the disulfide forms of beta-amino thiols are efficient prodrugs of aminopeptidase N inhibitors capable of crossing the blood-brain barrier.


The significance of low density microfilaraemia in the transmission of lymphatic filarial parasites. Low density microfilaraemia (mf) is a density of circulating mf which is often undetected by standard survey techniques; it occurs naturally, after anti-filarial drug administration and after vector control. Its occurrence in human populations is closely related to the observed mf frequency distributions in them, and it is an important cause of underestimation of mf prevalence rates in epidemiological surveys. In the present paper it is defined quantitatively as a count of less than 4 mf 20 microliters-1 of capillary blood or less than 30 mf ml-1 of venous blood. Detection of low intensity transmission of parasites is difficult; detection by clinical, entomological or immunological methods may be more sensitive than the usually employed parasitological techniques, due to the extreme inefficiency of the transmission process. Mosquito vectors of filariasis ingest and develop low density mf readily; since they exhibit limitation or proportionality, Aedes, Culex and Mansonia spp. vectors do this more efficiently than Anopheles spp. which exhibit facilitation. Field studies indicate that low level microfilaraemia can initiate a resumption of transmission after very efficient control programmes where Aedes spp. are vectors, whereas eradication has been achieved in areas of Anopheles transmission by levels of vector control which fall far short of eradicating malaria. The situation in the extensive endemic areas where Culex spp. are vectors is less clear, and should be a research priority.


Transkinetoplastidy--a novel phenomenon involving bulk alterations of mitochondrion-kinetoplast DNA of a trypanosomatid protozoan. Dramatic and consistent changes of mitochondria or kinetoplast DNA (kDNA) were observed in certain variants of Leishmania amazonensis (A variants) selected in vitro for arsenite-resistance. This was found initially by comparing different lots of wild-type cells and their respective A variants resistant to 30 microM arsenite. The kDNAs isolated from these two groups had different restriction patterns and hybridized poorly to each other, whereas those from different lots within each of the two groups were identical. Hybridization data showed an overall identity of less than 10(-3) between total kDNAs of the two groups. This difference was further examined in three independent series of variants, which were selected from three different clones for resistance to graded concentrations of arsenite (5-50 microM). In all three series, their kDNAs were found to change abruptly in an identical pattern at a late step of the selection process, i.e., A variants resistant to 15 microM or 30 microM arsenite. There was no apparent loss of kDNA in the process. Most of the changes observed appear to involve a shift in either the dominance or the copy number of different minicircle subclasses. Surprisingly, the kDNAs of tunicamycin-resistant variants (T variants) were also found to undergo similar changes. Genetic changes previously described in both A and T variants are limited to their nuclei. Namely, different chromosomal regions are amplified to produce large DNA circles which are responsible for the drug-resistant phenotypes. Interestingly, other arsenite-resistant clones without such chromosomal DNA amplification (A' variants) had kDNA of the wild-type pattern. The profound changes of kDNA observed are unprecedented. We propose the term "transkinetoplastidy" for this phenomenon to distinguish it from dyskinetoplastidy or the loss of kDNA described previously in trypanosomatid protozoa. This phenomenon is discussed with respect to the possible mechanisms of its generation, regulation and relation to the drug-resistant phenotypes.


Ultrastructure of Tricornia muhezae N. G., n. sp. (Microspora, Thelohaniidae), a parasite of Mansonia africana (Diptera: Culicidae) from Tanzania. Collections of Mansonia africana mosquito larvae were made at one site in N.E. Tanzania in 1985 and 1987 and from two additional sites, both within about 2 mi of the original one in 1987. An octosporous microsporidian, present at all three sites, was found in both years infecting from 7 to 22% of larvae. Spores (stained in Giemsa) measured 3.0 microns +/- 0.25 microns x 2.25 microns +/- 0.26 microns. Ultrastructurally, spores were seen to have an anterior rim surrounding a depressed area where the endospore was at its thinnest. In transmission electron microscopy section, the rim appeared as two processes into which all layers of the wall extended. At the posterior end all layers of the wall extended into a simple knob-like structure which could be interpreted as a section through a crest running longitudinally around the spore. The polar filament was anisofilar, with two anterior coils of greater diameter than the three posterior coils. Although most closely resembling the genera Amblyspora and Parathelohania in the family Thelohaniidae, the species in M. africana differs from the former, which has oval spores, broadly rounded at the ends, and from the latter, which has a prominent, ridged posterior extension to the spores. The new species has been placed in a new genus and the name Tricornia muhezae proposed.


ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, can infect both CD4+ and CD8+ T cells but requires CD4+ T cells in order to cause paralysis and immunodeficiency. When neonatal FVB/N mice were inoculated with ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, they developed a progressive bilateral hindlimb paralysis and immunodeficiency leading to death 4 to 6 weeks after inoculation. T lymphocytes have been shown to be primarily responsible for this ts1-induced syndrome. Here we compare the role played by each subset of T lymphocytes, i.e., CD4+ and CD8+ T cells, in disease development. Mice were depleted of a specific subset for the first 10 days of their lives by using either anti-CD4 or anti-CD8 monoclonal antibodies in vivo. Disease development in these mice was then monitored. Depletion of CD4+ T cells significantly attenuated the ts1-induced syndrome: virus replication was decreased, disease latency was extended, and death was prevented in 60% of the mice. Similar treatment with anti-CD8 antibody had almost no effect on disease progression. However, when depletion was begun 2 weeks after neonatal ts1 inoculation, CD4+ T cell depletion did not affect disease development. ts1 infected CD4+ and CD8+ T lymphocytes equally well in vivo, as shown by flow cytometric analysis, but virus replication was restricted primarily to the CD4+ subset of T cells, as found by in vitro assay. Hence, CD4+ T lymphocytes play an important role in the development of ts1-induced paralysis and immunodeficiency. The mechanism of this CD4+ T-cell-mediated disease production by ts1 is not clear; however, increased replication of ts1 in the CD4+ T cells, especially in the early stages of the disease, seems to play a crucial role.


Specific lysis of targets expressing varicella-zoster virus gpI or gpIV by CD4+ human T-cell clones. Varicella-zoster virus (VZV)-specific CD4-positive T cells are known to lyse targets expressing VZV antigen, but little is known of the glycoprotein specificity or phenotype of these cells. To test the ability of T cells to distinguish between gpI and gpIV (which share an antibody-defined epitope), we prepared clones from blood from four healthy individuals by limiting dilution. Among 68 T-cell clones from four donors which were VZV specific in tests of proliferation, 30 lysed autologous Epstein-Barr virus-transformed lymphoblasts which had been superinfected with a recombinant vaccinia virus which included the whole VZV gpI sequence. These clones were characterized as major histocompatibility complex class II restricted by inhibition of their cytotoxicity with HLA-DR and CD4 monoclonal antibodies. Twenty-one clones lysed targets expressing gpIV. Fifteen of these clones lysed targets expressing gpI and gpIV. Four clones with gpI-gpIV specificity were examined in detail, and their dual specificity was confirmed by cold target inhibition. These four clones failed to kill target cells infected with a mutant gpIV recombinant vaccinia virus from which amino acid residues 212 to 354 had been deleted. This region includes one of the two gpIV decapeptides which have 50% homology with amino acids 111 to 121 of gpI. Our data confirm that T-cell-receptor-associated structures are required for specific lysis of VZV targets and indicate that (i) gpI-specific CD4 cytotoxic T cells outnumber gpIV-specific T cells in blood and (ii) 50% of gpI-specific T-cell clones also lyse gpIV-expressing targets.


Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes. The lentiviruses of sheep, goats, and horses cause chronic multiorgan disease in which macrophages are highly permissive for viral replication. Monocytes, which mature into macrophages, are thought to be latently infected with lentivirus, but the extent to which other leukocytes are infected is unknown. Dendritic cells have not been studied separately from monocytes and T-cell subsets have not been examined in previous attempts to identify infected cells in peripheral blood mononuclear cells (PBMC). We found no evidence of T-cell tropism using an animal-passaged, pathogenic ovine lentivirus. Phytohemagglutinin-stimulated infectious PBMC produced 20-fold less virus than differentiated macrophages, and cocultivation of infectious PBMC with fresh, uninfected phytohemagglutinin blasts did not facilitate virus replication. Furthermore, central lymph cells, the best in vivo source of purified lymphocytes, lacked virus and did not yield virus upon in vitro cultivation. In contrast, cultivated blood-derived macrophages were highly permissive for viral replication. To identify the latently infected PBMC, PBMC from infected sheep were selectively depleted of monocytes and B cells by passage over nylon wool and then of nonadherent cells bearing CD4, CD8, T19, gamma delta T-cell receptor, CD45RA, or major histocompatibility complex class II antigens by panning. Removal of adherent monocytes and B cells or of adherent cells and the three major T-cell subsets (CD4+, CD8+, T19+) did not decrease the infectivity of PBMC. The richest sources of infected cells in fresh PBMC were CD45RA+ and major histocompatibility complex class II+ nonadherent cells, which are three characteristics of dendritic cells. Thus, the dendritic cell, and not the monocyte or the CD4+ cell, is probably the predominant infected cell type in blood.


Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones. In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.


Human herpesvirus 7 is a constitutive inhabitant of adult human saliva. We report the frequent isolation of human herpesvirus 7 from the saliva of healthy adults. Virus isolates recovered from different individuals exhibited minimal restriction enzyme polymorphism, which was mostly confined to heterogeneous (het) sequences in the genome. DNAs of isolates recovered from the same individual over a period of several months showed the same characteristic het fragments, indicating the stability of the het sequences upon virus replication and shedding in vivo. In contrast to the results of previous reports, human herpesvirus 6, the causative agent of roseola infantum, could not be isolated from the saliva specimens, raising questions regarding oral transmission of human herpesvirus 6 and human herpesvirus 7 to young children.


[Autonomic nervous function and plasma catecholamine levels of perioperative patients treated with antipsychotic drugs]. The RR intervals on ECG and the coefficient of variation of R-R interval (CV) and perioperative plasma catecholamine levels were measured in patients receiving long-term administration of antipsychotic drugs. CV was significantly reduced (P less than 0.05) in patients on antipsychotic drugs. Preoperative plasma catecholamine levels increased significantly and elevation of plasma catecholamine levels on the first postoperative day was less than that of the control group. These findings suggest that the patients on antipsychotic drugs have disturbed autonomic nervous function. Therefore, a careful perioperative care is mandatory.