Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype. The retinoblastoma-predisposition gene, RB1, segregates as an autosomal dominant trait with high (90%) penetrance. Certain families, however, show an unusual low-penetrance phenotype with many individuals being unaffected, unilaterally affected, or with evidence of spontaneously regressed tumors. We have used single-strand conformation polymorphism analysis and PCR sequencing to study two such families. Mutations were found in exon 20 of RB1 in both cases. In one family a C----T transition in codon 661 converts an arginine (CGG) to a tryptophan (TGG) codon. In this family, incomplete penetrance and mild phenotypic expression were observed in virtually all patients, possibly indicating that single amino acid changes may modify protein structure/function such that tumorigenesis is not inevitable. In the second family the mutation in codon 675 is a G----T transversion that converts a glutamine (GAA) to a stop (TAA) codon. However, this mutation also occurs near a potential cryptic splice acceptor site, raising the possibility of alternative splicing resulting in a less severely disrupted protein.


Genetic, cytogenetic, and molecular analyses of mutations induced by melphalan demonstrate high frequencies of heritable deletions and other rearrangements from exposure of postspermatogonial stages of the mouse. Specific-locus experiments have previously shown melphalan to be mutagenic in all male germ-cell stages tested and particularly so in early spermatids. All but 2 of 24 specific-locus mutations recovered were tested genetically, cytogenetically, and/or molecularly. At least 12 of 15 tested mutations recovered from postspermatogonial stages but only 1 of 7 mutations recovered from stem-cell or differentiating spermatogonia gave evidence of being deletions or other rearrangements. Melphalan-induced mutations, thus, confirm the pattern of dependence of mutation structure on germ-cell stage that had been shown earlier for other chemicals. Results of the present investigation illustrate the capabilities of combined genetic, cytogenetic, and molecular analyses for characterizing the nature of specific-locus mutations. Fine-structure molecular mapping of long regions surrounding specific loci has been greatly facilitated by the availability of genetic reagents (particularly, deletion complexes) generated in specific-locus experiments over the course of decades. Reciprocally, this mapping permits increasingly detailed characterization of the nature of lesions induced by mutagenic exposures of germ cells, adding great powers for qualitative analysis of mutations to the specific-locus test. Cytogenetic and genetic investigations also provide evidence on lesion type, especially for loci at which mutations cannot yet be analyzed molecularly. Melphalan, like chlorambucil, can generate many mutations, a high proportion of which are deletions and other rearrangements, making this chemical valuable for generating mutations (at any locus) amenable to molecular access.


The LIM family transcription factor Isl-1 requires cAMP response element binding protein to promote somatostatin expression in pancreatic islet cells. Many eukaryotic genes are regulated by cAMP through a conserved cAMP response element (CRE). Here we show that, in the pancreatic islet cell line Tu6, a well-characterized CRE in the somatostatin gene does not provide cAMP responsiveness but functions as an essential element for its basal activity. DNA-binding and functional analyses indicate that the cAMP-responsive factor CREB regulates somatostatin expression in these cells without requirement for phosphorylation at the protein kinase A-regulated Ser-133 phosphorylation site. In addition to the CRE site, cell-specific expression of the somatostatin gene requires a second promoter element, which binds the recently characterized LIM family protein Isl-1. Thus, Isl-1 and CREB appear to synergize on the somatostatin promoter to stimulate high-level expression in Tu6 cells. The ability of CREB to function in a phosphorylation-independent manner suggests a mechanism by which this protein can regulate gene transcription.


Murine Hox-1.11 homeobox gene structure and expression. The Hox-1.11 gene encodes a protein 372 amino acid residues long that contains a conserved pentapeptide, a homeodomain, and an acidic region. The amino acid sequence of the homeodomain of Hox-1.11 is identical to that of Hox-2.8, and the N-terminal and C-terminal regions of Hox-1.11 are similar to those of human HOX2H, which is the equivalent of murine Hox-2.8. The Hox-1.11 gene was shown to reside on murine chromosome 6, which contains the Hox-1 cluster of homeobox genes. One species of Hox-1.11 poly(A)+ RNA approximately 1.7 kb long was detected in mouse embryos, which is most abundant in 12-day-old embryos and progressively decreases during further embryonic development. The most anterior expression of Hox-1.11 poly(A)+ RNA in 12- to 14-day-old mouse embryos was shown by in situ hybridization to be in the mid and posterior hindbrain. Hox-1.11 poly(A)+ RNA also is expressed in the VII and VIII cranial ganglia, spinal cord, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine.


Detection of mutant Ha-ras genes in chemically initiated mouse skin epidermis before the development of benign tumors. An activated Ha-ras oncogene has been consistently found in chemically initiated benign and malignant mouse skin tumors, and an activated ras oncogene has been shown to initiate the process of mouse skin carcinogenesis. However, the exact timing of mutational activation of the Ha-ras gene relative to application of the chemical carcinogen is not known. A sensitive mutation-specific PCR technique was used to experimentally address the timing of Ha-ras gene mutational activation. This technique can detect mutant Ha-ras alleles in the presence of a very large excess of normal ras alleles. Activated Ha-ras genes with 61st codon A----T mutations were found in the epidermis of mice 1 week after topical initiation with 7,12-dimethylbenz[a]anthracene or urethane by using this assay. These results were confirmed by Xba I restriction fragment length polymorphism analysis and direct DNA sequencing. One week after initiation is 1-2 months before the appearance of benign papillomas that harbor activated Ha-ras oncogenes when the initiated mice are promoted with the tumor promoter phorbol 12-myristate 13-acetate. Our data support the hypothesis that initiated epidermal cells containing an activated Ha-ras gene can remain dormant in the skin until a tumor promoter induces regenerative hyperplasia that allows for outgrowth of these cells with an activated ras oncogene to give rise to a benign papilloma.


Genetic and developmental factors in the olfactory response of Drosophila melanogaster larvae to alcohols. Olfactory responses of Drosophila melanogaster larvae to a homologous series of primary alcohols (methanol ... decanol) were tested. Alcohols at either extreme of the chain lengths studied (methanol, ethanol and decanol) evoked no significant responses. Heptanol and nonanol both produced dose-independent responses, larvae being attracted to heptanol and repulsed by nonanol. The remaining alcohols elicited dose-related attractive responses. Responses to hexanol and nonanol decline with increasing larval age. Genetic differences were found for the response to heptanol, with larvae from a Japanese strain, Katsunuma, being indifferent to this substance. Chromosome exchange revealed that a major factor involved in the response to heptanol is located on chromosome II; factors on chromosome III quantitatively modulate this response. Three mutant strains were isolated following EMS mutagenesis of chromosome III. These three strains, IndifferentA, IndifferentB and IndifferentC, show incomplete or total anosmia when stimulated with nonanol. Adult flies from these strains show similar effects. IndifferenB and C strains are dominant over the Canton-S control strain; the IndifferentA strain shows semi-dominance. Results are discussed in the light of the ecology of Drosophila larvae and the relation between olfactory stimulus and receptor conformation and number.


Cell-specific contact selects transmitter responses in an identified leech neuron. Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.


Genomes of diploblastic organisms contain homeoboxes: sequence of eveC, an even-skipped homologue from the cnidarian Acropora formosa. We report the nucleotide sequence of eveC, a cnidarian eve-class homeobox; this is the first homeobox to be identified in any diploblastic organism, and is only the second eve-class in an invertebrate. Similarity between the predicted amino acid sequence of the eveC homeodomain and its insect and vertebrate equivalents was approximately 75-80% but, in the case of eveC, a role in segmentation can be ruled out. Our findings thus support the 'co-option' hypothesis: homeoboxes were an early feature of metazoan genomes, corresponding to the DNA-binding domains of more general transcription factors.


Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice. In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.


Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons. Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.


Effects of pattern element density upon displacement limits for motion detection in random binary luminance patterns. The upper motion displacement threshold (Dmax) was determined with two-frame motion sequences of random binary luminance patterns, over a range of pattern element sizes and densities. Dmax was little affected by density at small element sizes (less than 5 arcmin), in agreement with previous reports. However, at larger element sizes (greater than 9 arcmin) Dmax increased as element density was reduced in the range 50-5%. We explain our findings by a model which takes into account spatial-frequency filtering prior to motion detection, and the effects of pattern density upon the statistics of random binary patterns. We also implicate the dependence of Dmax upon the contrast energy of the elements in broadband patterns, and provide a direct demonstration that Dmax is contrast limited over a wide range of pattern contrasts (72-2.5%). Previous reports that Dmax is independent of density should be modified to take into account the complex effects of density upon the statistics of random patterns, and the existence of physiological filtering prior to motion detection.


Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes. Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.


Second messenger imbalance hypothesis of schizophrenia. Based on the results of studies on intracellular signaling in platelets of schizophrenics, an imbalance of the second messenger system is proposed: Diacylglycerol (DG), which activates protein kinase C (PKC), was increased, while adenylate cyclase (AC)-cAMP function was decreased. It is proposed that the increased DG/PKC function may entail a decrease in inositol 1,4,5-trisphosphate (IP3)/Ca2+ function and lowering of phosphoinositide turnover. If such a pathological intracellular signaling takes place in the brain, it may cause a distorted balance of protein activation via phosphorylation in neurons, resulting in some of the deficits of schizophrenia. Neuroleptics have been reported to antagonize the above-mentioned pathological processes of intracellular signaling. The imbalance hypothesis of the DG/PKC pathway and AC-cAMP pathway is not inconsistent with the dopamine hypothesis of schizophrenia, because dopamine receptor stimulation is indirectly related to reduction in IP3/Ca2+ function and lowering of phosphoinositide metabolism.


Increased turnover of platelet phosphatidylinositol in schizophrenia. The potential role of receptor-stimulated phosphatidylinositol (PI) hydrolysis in a signal transduction mechanism has been increasingly recognized. Earlier studies have suggested a defect in alpha-adrenergic receptor function in the platelets of schizophrenic patients. Little is known, however, about the mechanisms for PI synthesis, breakdown, and regulation in schizophrenia. The present study was undertaken to investigate the metabolic turnover of inositol phospholipids and inositol phosphates by incorporation of [3H]myoinositol or [32P]orthophosphate into resting and activated platelets of normal controls and schizophrenic patients with and without neuroleptic treatment. After 5 h incubation at 37 degrees C, the majority of [3H]myoinositol was incorporated into platelet PI. Following thrombin-induced platelet activation, there was rapid formation of 3H-labeled inositol phosphates (IPs) with inositol monophosphate (IP1) being the most abundant product. The thrombin-induced formation of platelet IPs was found significantly higher in both haloperidol-stabilized and drug-free schizophrenics than in normal control subjects. When platelets were prelabeled with [32P]orthophosphates, thrombin-induced formation of phosphatidic acid (PA) was also significantly higher in haloperidol-stabilized schizophrenics than in normal controls. It is thought that thrombin-induced platelet activation is mediated through hydrolysis of polyphosphoinositides (poly-PI). The present data thus may reflect an increased signal transduction in schizophrenia, which is mediated through neuroleptic-regulated inositol phospholipid hydrolysis.


Inositol phospholipid turnover in platelets of schizophrenic patients. Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.


Administration of AP5, a glutamate antagonist, unmasks glycine analgesic actions in the rat. The effect of intrathecal (IT) injection of glycine alone or in combination with 2-amino-5-phosphonopentanoate (AP5) on two nociceptive tests--the vocalization threshold to tail-shock (VTTS) and the tail-flick latency (TFL)--was studied in ovariectomized Sprague-Dawley rats. IT injection of 400 micrograms glycine induced a nonsignificant decrease, that is, in comparison with saline, in both nociceptive thresholds. IT AP5 (10 micrograms) provoked a slight but significant increase in both nociceptive thresholds within the first 15 min postinjection. Combination of both glycine (400 micrograms) and AP5 (10 micrograms) produced marked and prolonged analgesia in both tests, which was significantly different from that obtained with AP5 alone. The results suggest that IT glycine acting through the strychnine-sensitive Gly1 receptor produces analgesia provided its effect on the Gly2 receptor linked to the NMDA receptor is prevented by an antagonist.


Reproducibility of time structure in motor activity of rats under nocturnal conditions. Computer pattern recognition systems for the study of spontaneous rat behavior have introduced a new analytical technique, the K functions, that expands the definition of experimentally induced changes in behavior. Such studies normally evaluate three measures, a measure of the number of initiations of specific behavioral acts, a measure of the total time of each act, and a measure of behavioral time structure, the K functions. Such measures have been shown to be very stable and reproducible among control rats observed under normal light conditions. This study examines the stability of results from these three measures as applied to nine different groups of control Sprague-Dawley male rats observed under red light during their normal nocturnal hours. All three measures provided stable and reproducible results, but the measure of time structure, the K function analysis, provided the greatest consistency, producing values that vary by only a few percent.


Differential effects of dopamine D2 agonist quinpirole upon the dorsal immobility response in rats. The effects of various dose levels of systemically injected quinpirole upon the dorsal immobility response (DIR) over a time course was investigated in male rats. A low dose of quinpirole (0.01 mg/kg) significantly attenuated the duration of the DIR following the 10-min interval, whereas the highest dose (1.0 mg/kg) had a biphasic effect so that at the 10-min interval the duration of the DIR was significantly potentiated and at the 60-min interval the duration of the DIR was significantly attenuated. The intermediate dose (0.1 mg/kg) had intermediate behavioral effects. The data support the growing evidence that quinpirole has differential effects upon behavior over time as a function of the dose levels. The present data were discussed in reference to presynaptic and postsynaptic dopamine D2 receptor theory.


Rapid and simultaneous assay of monoamine neurotransmitters and their metabolites in discrete brain areas of mice by HPLC with coulometric detection. For simultaneous assay of the three monoamine neurotransmitters, norepinephrine, dopamine, and serotonin, and four respective metabolites in brain tissue, a rapid and simple method using high-performance liquid chromatography with coulometric detection is described. Because the present method permits the determination of these target substrates within 10 min or less in one chromatographic run, 150 samples can be analyzed using an autosampler and an integrator in a 24-h period. Within-run coefficients of variation for the target substrates in the standard solution and the whole brain sample were less than 3% and 2% (n = 40), respectively. The quantitative detection limits were 0.01-0.1 pmol. The present procedure was applied to measure the target substrates in several discrete brain areas in mice.


Symptomatology and electrodermal activity as predictors of neuroleptic response in young male schizophrenic inpatients. This study examined whether the response to treatment with neuroleptic medication in 21 schizophrenic patients could be predicted from symptomatology, electrodermal activity, and premorbid adjustment. Positive symptoms and high levels of electrodermal activity were associated with a good response to conventional neuroleptic drugs. However, multivariate analysis indicated that symptomatology was the only independent predictor of treatment response.


[Current perspectives in bone marrow transplantation]. Peripheral blood and umbilical cord blood are promising sources of hemopoietic stem cells for autologous as well as allogeneic transplantation. The nature of the progenitors responsible for early marrow reconstitution after transplantation on one hand and for permanent reconstitution on the other hand is getting more precise. Features of the cells responsible for GVHD and of those responsible for GVL effect (graft versus leukemia effect) will soon allow a distinct monitoring of these two activities. Molecular biology will soon permit genetic labelling of the graft and thereby bring more precision for its follow-up and modulation in vivo in the post-graft period. Correction of an autologous transplant by introduction of the adequate gene in the hematopoietic stem cells in case of genetic anomaly at this level, should in the future, as suggested by animal studies, allow permanent correction of genetic disorders in patients autografted according to such a procedure. Finally, introduction of procedure like antisense nucleotides against DNA regions responsible for the malignant proliferation of tumoral cell, could extend the therapeutic possibilities of autograft.


Gastroprotective and antisecretory effects of ebrotidine. This study was designed to assess the gastroprotective and secretory effects of ebrotidine, a novel H2-receptor antagonist, in humans. Two groups (A and B) of male subjects with normal gastric mucosa were used. Group A (six subjects) was treated for 3 days with either ebrotidine or placebo in a randomized, crossover study, and on the 4th day 100 ml of 50% ethanol was sprayed on the mucosa via an endoscope. Pretreatment with ebrotidine significantly reduced the endoscopic score of mucosal damage and deep hemorrhagic lesions caused by ethanol as compared with those in placebo-treated subjects. In group B (six subjects) the 24-h pH-metry was assessed with an intraluminal pH electrode placed in the gastric corpus and connected to portable recording apparatus. A single oral dose of ebrotidine (800 mg) caused a significant reduction in circadian acidity and resulted in a marked and significant inhibition of acid secretion for about 6 h on administration. We conclude that ebrotidine is highly effective as a gastroprotective agent, and as an H2-receptor antagonist shows a potent inhibitory effect on gastric acid secretion in humans.


Somatostatin cells in the oxyntic mucosa of hypo- or hypergastrinemic rats. The present report describes the long-term effects of antrectomy, antrum exclusion, portacaval shunt, omeprazole treatment, or the combination of omeprazole treatment and portacaval shunt on the number and density of somatostatin cells in the oxyntic mucosa of the rat. Antrectomy, which is associated with hypogastrinemia, raised the number and density of the somatostatin cells, whereas antrum exclusion and omeprazole treatment, which are associated with hypergastrinemia, reduced the number and density of the somatostatin cells. Portacaval shunt, which is associated with hypogastrinemia, increased both the number and the density. Omeprazole treatment of portacaval--shunted rats suppressed or even reversed the somatostatin cell hyperplasia after portacaval shunt alone. From these findings it is unlikely that gastrin stimulates the proliferation of somatostatin cells in the oxyntic mucosa. In fact, there seems to be an inverse relationship between the serum gastrin concentration and the somatostatin cell number.


Programmed death of T cells in HIV-1 infection. In human immunodeficiency virus (HIV) infection, functional defects and deletion of antigen-reactive T cells are more frequent than can be explained by direct viral infection. On culturing, both CD4+ and CD8+ T cells from asymptomatic HIV-infected individuals died as a result of programmed cell death (apoptosis). Apoptosis was enhanced by activation with CD3 antibodies. Programmed cell death, associated with impaired T cell reactivity, may thus be responsible for the deletion of reactive T cells that contributes to HIV-induced immunodeficiency.


Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8+ cytomegalovirus-specific CTL responses.


Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ T cells. The paucity of virus-laden CD4+ cells in individuals infected with human immunodeficiency virus type-1 (HIV-1) contrasts with the greatly reduced numbers and function of these lymphocytes. A pathway is described whereby dendritic cells carry HIV-1 to uninfected T cells, amplifying the cytopathic effects of small amounts of virus. After exposure to HIV-1, dendritic cells continue to present superantigens and antigens, forming clusters with T cells that are driven to replicate. Infection of the dendritic cells cannot be detected, but the clustered T cells form syncytia, release virions, and die. Carriage of HIV-1 by dendritic cells may facilitate the lysis and loss of antigen specific CD4+ T cells in acquired immunodeficiency syndrome.


A light-microscopic immunocytochemical study of the endocrine pancreas in the Australian fat-tailed dunnart (Sminthopsis crassicaudata). The endocrine pancreas of the Australian fat-tailed dunnart (Sminthopsis crassicaudata) was investigated by means of immunocytochemistry using the avidin-biotin-peroxidase technique. This was a light microscopic study using this established technique and has not been previously investigated. Serial paraffin sections were stained individually with primary antibodies for anti-porcine glucagon, anti-beef pork insulin, anti-human somatostatin, and anti-avian pancreatic polypeptide (APP), anti-bovine pancreatic polypeptide (BPP), anti-serotonin, anti-porcine motilin, showing the same islet. Cells immunoreactive to porcine glucagon, porcine insulin, human somatostatin, APP, BPP were found in endocrine islets, but BPP and APP also appear to be scattered amidst the exocrine portion. Immunoreactive cells were not observed with serotonin and anti-porcine motilin. All controls were negative. These results in the dunnart pancreas has shown four types of pancreatic endocrine cells. It has also shown that the structure of PP may more closely resemble BPP than APP. This study can be related to studies in echidnas (Tachyglossus aculeatus) and Australian possum (Trichosurus vulpecula). This is a part of an immunocytochemical study investigating the endocrine pancreas in Australian mammals.


Mucocutaneous abnormalities predicted by lymphocyte counts in patients infected with the human immunodeficiency virus. Abnormalities of the skin are frequent and troubling problems for patients infected with the human immunodeficiency virus (HIV). A number of studies have assessed the frequency and severity of diseases of the skin and mucous membranes reported from other centers, but relationships between dermatologic signs and symptoms and either the lymphocyte count or the helper T-lymphocyte count have been infrequently noted. In a prospective study of 6 months' duration, one of us (A.F.) examined and questioned 61 HIV-seropositive patients at our infectious disease clinic. We found a significant association between the number and severity of cutaneous abnormalities and the helper T-cell (CD4) count. A trend toward significance was also shown between advanced HIV-disease status or decreased CD4 counts and pruritus. Our findings suggest that both the peripheral blood lymphocyte count and the helper T-cell count are predictive of the frequency, severity, and symptoms of skin diseases.


[Vascular complications in reconstructive surgery of the foot]. Even when we assess accurately the preoperative vascular damage of the blood vessel we intend to use for reconstruction, difficulties may arise during operation which cannot be resolved by routine methods. Microvascular surgery is an important part of many modern reconstruction procedures which can be used during reconstruction of the extremity. As an example the authors describe injuries of the left foot involving loos in a 41-year-old patient who was knocked down by a taxi. The defect denuded the bone and loss of soft tissues of the heel called for free transfer of a vascularized flap. During operation venous thrombosis and subsequently arterial thrombosis developed. The use of an interposed vein helped to ensure a sufficient blood supply to the transferred tissue. The left foot healed well.


Use of internal mammary artery in radiation-induced coronary artery disease. In 3 patients who had been subjected to mediastinal irradiation for breast cancer, the internal mammary artery was used as a conduit for myocardial revascularization. Intraoperatively, in all patients the internal mammary artery exhibited excellent blood flow and except for mild adhesions between the pericardium and epicardium no unusual technical problems were encountered. Preoperative assessment of the internal mammary artery by angiography is recommended in patients with radiation-induced coronary artery disease who are scheduled to undergo myocardial revascularization with intended use of the internal mammary artery.


Blockade of N-methyl-D-aspartate receptors prevents cyanide-induced neuronal injury in primary hippocampal cultures. Cyanide-induced alterations of cytosolic calcium levels and cytotoxicity were examined in primary cultures of rat hippocampus. Cytosolic free Ca2+ ([Ca2+]i) levels were measured in hippocampal neurons using the fluorescent probe, fura 2. A concentration-dependent rise in [Ca2+]i occurred rapidly following exposure of cells to 0.5-10 mM NaCN. In normal medium (1.3 mM Ca2+), 2 mM NaCN produced an increase in [Ca2+]i (172 +/- 27% of control), 45 sec following exposure. Ca2+ elevation produced by NaCN was blocked by removal of Ca2+ from the external medium or by pretreatment with the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovalerate (APV). The cytotoxicity of cyanide, assessed by measuring the efflux of lactate dehydrogenase, was blocked by APV. These results indicate that in hippocampal neurons, cytosolic Ca2+ accumulation induced by cyanide originates from the extracellular compartment and the NMDA receptor ionophore is a significant route for Ca2+ entry. It is proposed that excitotoxic mechanisms may contribute to altered neuronal homeostasis and injury associated with cyanide.


The effects of liver denervation on the regulation of hepatic biliary secretion. Effects of liver denervation on bile formation were studied in eight dogs prepared with chronic biliary fistulas. The animals were studied in the basal state, after feeding, and during infusion of glucagon 50 ng/kg/min, secretin 2 U/kg/hr, or somatostatin 200 ng/kg/min. After this first set of experiments the animals underwent a total hepatic denervation that consisted of section of the hepatic ligaments and a careful dissection of the portal vein, hepatic artery, and common duct with stripping of all the surrounding connective tissue and topical application of phenol. The above experiments were then repeated. Denervation did not modify bile flow, or bile salts, cholesterol, or phospholipid concentration or output. Biliary response to glucagon and secretin was similar before and after denervation. Somatostatin had an anticholerectic effect in both intact and denervated animals, but significantly reduced bile salt output only in the intact dogs. Feeding had a choleretic effect pre- and postdenervation, and the infusion of somatostatin following feeding decreased bile flow to the same degree before and after denervation. In the intact animals the output of all three biliary lipids was reduced by somatostatin after feeding but they were unaffected by somatostatin after denervation. Moreover, cholesterol and phospholipid outputs were stable after feeding in intact animals, but significantly decreased after denervation. 14C-erythritol clearance studies indicated no change in the canalicular component of bile flow with denervation, except again during somatostatin suppression of feeding. These data indicate that basal bile flow is normal after denervation but that innervation may play an important role in the modulation of responses to somatostatin and more complex stimuli such as feeding.


The distribution of the CDW52 molecule on blood cells and characterization of its involvement in T cell activation. The O97 mouse mAb was used to define, together with the CAMPATH-1 mAb series, the CDw52 in the course of the Fourth International Workshop on Human Leucocyte Differentiation Antigens. O97 and CAMPATH-1M produce full competitive inhibition and react with an epitope dependent on O-linked sugar residues. The two mAb, however, display significant differences in reactivity with leukocyte populations--in fact the reactivity of O97, which also exerts a powerful cytotoxic effect with human C', is similar to mAb from the CAM-PATH-1 series having the broadest one. Noteworthy, O97 spares PBL, NK, and LAK precursors, permitting an easy purification of these cells; O97 is able to kill long-term colony-forming cells in bone marrow culture and characteristically reacts, in contrast to CAMPATH-1M, with erythrocytes. Western blotting has revealed a strikingly different molecular size on red cells, since CDw52 mAb revealed a broad band ranging from 6-16 kDa, instead of the 21-28 kDa revealed from leukocyte extracts. In agreement with immunofluorescence data, O97 revealed abundant material from red cells in Western blotting, while reactivity of CAMPATH-1M was very faint. Overall, these results show that distinct molecular forms of the CDw52 molecules are present on different blood cells. We have also characterized an activation signal that can be delivered to T cells via the CDw52 molecule by CAMPATH-1M but not by O97. This is an accessory signal that can complement a primary activation signal delivered via the CD2 pathway but not via the CD3-TcR pathway. This is similar to the effects obtained with the CD45R (2H4) mAb, 2H4 and CAMPATH-1M mAb having additive effects on T cell activation via CD2.


[Health-economic aspects of newer anti-ulcer drugs]. Peptic ulceration is a common condition and is associated with considerable expense. Introduction of H2-blockers in the latter part of the nineteen seventies offered a new and effective therapeutic alternative for the patient with chronic peptic ulceration. On the basis of a review of the literature, the present authors have attempted to assess the consequences of the introduction of H2-blockers for the expenses of peptic ulceration. Introduction of medication is found to have reduced the expenses involved in peptic ulceration on account of elective operations but on account of the increased costs of medication, the total direct costs involved in the treatment of peptic ulceration have possibly increased after the introduction of the medication. Attempts are made to compare this with the reduction in the indirect costs achieved by reduction in loss of production involved by fewer early retirals and sick leaves on account of peptic ulceration, fewer deaths connected with peptic ulceration among young persons and, in general, improved quality of life for patients with peptic ulceration although it is difficult to provide a valid estimation of the savings involved.


Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment. Interleukin-2 (IL-2) treatment of cells and generation of non-major histocompatibility complex (MHC)-restricted cytotoxic cells from peripheral blood mononuclear leukocytes (PBML) was studied. Effector-target conjugate assays demonstrated that bovine PBML bound but did not lyse K562, HL60S and HL60R cells unless activated with IL-2. The magnitude of IL-2-activated killing of tumor cells as well as the magnitude of antibody-dependent cellular cytotoxicity depended on the IL-2 concentration. A short treatment (12-18 h) of effector cells with IL-2 was sufficient for development of cytotoxic activity. Withdrawal of IL-2 from the culture resulted in a reduction of cytotoxic activity that could be restored by further addition of IL-2. Cytotoxic activity of IL-2-activated populations obtained after nylon wool or Sephadex G-10 passage, and Percoll gradient centrifugation of PBML suggests that lymphokine-activated killer (LAK) cell activity in PBML is mainly mediated by a non-adherent lymphocyte lacking markers for B-cells. Positive and negative selection experiments using cell sorting confirmed these findings and demonstrated that the cell responsible for LAK cell activity in cattle belongs to a non-monocyte, non-B, CD2+ lymphocyte population. Furthermore, cytotoxic activity could not be generated in CD2+ populations enriched for cells expressing molecules equivalent to human and murine CD4 and CD8. These findings suggest that effector cells mediating non MHC-restricted cytotoxicity in cattle prevail in a population bearing a CD2+, CD4-, CD8- phenotype and that this population depends on the continuous presence of IL-2 for optimal cytotoxic function.


Leucocyte adhesion protein deficiency in Irish setter dogs. Investigation of 12 Irish setter puppies from six litters with severe recurrent infections, neutrophilia and low body weight revealed a leucocyte adhesion protein deficiency with a total lack of CD11b and CD18. Their neutrophil function was severely impaired with a totally absent capacity to ingest C3b-opsonized particles, a significantly impaired capacity to ingest IgG-opsonized particles and significantly diminished adherence to nylon wool when compared with neutrophils from healthy control dogs. The chemiluminescence of patient neutrophils activated by C3b-opsonized particles was, consequently, significantly decreased compared with that of control neutrophils, while the respiratory burst assayed by phorbolmyristate acid (PMA) stimulated nitroblue tetrazolium (NBT)-reduction was normal in the patient group. Random migration and chemotactic responses of patient and control neutrophils, were similar. The etiology, pathogenesis and clinical manifestations of the Irish setter leucocyte adhesion deficiency were similar to that of the leucocyte adhesion deficiency in humans.


[Multicenter study of isradipine in the treatment of hypertension]. MIS is a one-year Multicentre Isradipine Study of the treatment of essential hypertension, in which participated seven centres in Czechoslovakia. The study comprised 144 patients with mild or medium severe hypertension. Isradipine belongs into the group of dihydropyridine derivatives with a high specific and low non-specific affinity to dihydropyridine binding sites of the L-type of calcium channels. After a four-week placebo period isradipine treatment (2.5 mg (1/2 tablet twice a day/was started. This dose increased to 5 mg (1 tablet twice a day) unless normalization of the diastolic pressure was achieved by a smaller dose. Monotherapy with isradipine normalized the diastolic pressure (less than 90 mmHg) in 44% of the hypertonic patients. 56% hypertonics where monotherapy with isradipine did not reduce the diastolic pressure below 90 mmHg were treated by a combination of isradipine and bopindolol. This group of patients had a significantly higher systolic and diastolic pressure, a higher number of erythrocytes and thrombocytes at the onset of the investigation. Addition of bopindolol to isradipine proved very effective. At the end of the one-year study 87% of the patients had a normal diastolic pressure. Isradipine as monotherapy and combined with bopindolol did not influence the metabolic risk factors of IHD and drug tolerance was very good.


The metabolism of imiloxan hydrochloride in healthy male volunteers. 1. The metabolism of imiloxan hydrochloride [(+-)-2-(1-ethyl-2-imidazoyl)methyl-1,4-benzodioxane hydrochloride], an alpha 2-adrenoceptor antagonist, was studied in four male volunteers given a 500 mg oral dose containing 0.48 MBq of the 14C-labelled material. Compound-related radioactivity was rapidly excreted chiefly in the urine within 24 h of dosing. 2. Metabolites derived by initial oxidation on either or both the benzodioxane and imidazoyl moieties followed by glucuronic acid and sulphate conjugation, and an N-glucuronide of imiloxan were tentatively identified in urine. 3. The major urinary metabolites, comprising some 37-41% of the dose, appeared to be +-2-(1-ethyl-2-imidazoyl)methyl-1,4-benzodioxane-6/7-sulphonic acid (19% of dose), [+-2-(1-ethyl-2-imidazoyl)methyl-1,4-benzodioxane- 6/7-ylium D-glucopyranoside]uronate (10-14% of dose), and a glucuronide conjugate of +-2-(1-ethyl-2-imidazoyl-4/5-hydroxy)methyl-1,4-benzodioxane (8% of dose).


A randomized placebo controlled trial on the effects of simvastatin, a HMG-CoA reductase inhibitor, on blood lipids and fibrinolytic parameters. Coronary artery disease is frequently associated with disturbed blood lipids and with a deficient blood fibrinolytic capacity. In order to investigate a possible link between hypercholesterolemia and hypofibrinolysis, we have investigated the effect of simvastatin, a HMG-CoA reductase inhibitor, on blood lipids and fibrinolytic parameters in a double blind, randomized, placebo controlled study design. Twenty-four male patients, aged between 42 and 65 years, with angiographically confirmed coronary artery disease, were selected from a series of 731 consecutive patients on the basis of a fasting serum cholesterol level of more than 250 mg/dl and a plasma PAI-1 level of more than 60 ng/ml. Patients were randomly assigned to 20 mg of simvastatin daily (Group I), or placebo (Group II), for four weeks, followed by doubling of the dose for another four weeks. Blood samples were obtained at baseline and at 4 and 8 weeks. Groups I and II did not differ significantly at baseline. As expected, simvastatin produced a significant reduction in serum levels of total cholesterol (33 +/- 12 and 36 +/- 12 percent, mean +/- SD, after 4 and 8 weeks respectively, p less than 0.001 vs baseline), LDL-cholesterol (36 +/- 5 and 43 +/- 6 percent respectively, p less than 0.001 vs baseline) and apolipoprotein-B (20 +/- 29 and 23 +/- 30 percent respectively, p less than 0.05 vs baseline), whereas these parameters did not change significantly in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)


Alfentanil combined with vecuronium or pancuronium: haemodynamic implications. Forty-two fit, anticholinergized patients, induced with thiopentone, received either vecuronium (V) or pancuronium (P) 0.1 mg/kg, followed by alfentanil 15 micrograms/kg. The mean heart rate in the Group V was significantly lower than that in the Group P. The difference, 10-15 bpm, appeared after alfentanil administration, and lasted for 5 min postintubation, when under N2O anaesthesia. The Group P patients maintained their arterial pressure closer to the preinduction level than did the Group V patients, but a statistically significant inter-group difference appeared only at two recording stages. Four Group V patients, contrasted to none of the Group P patients (P less than 0.05), were put in head-down tilt, or were given atropine, and/or etilephrine for an undue decrease in arterial pressure. Compared to vecuronium, pancuronium increased heart rate, and protected from arterial hypotension, when combined with low-dose alfentanil.


In situ characterization of mononuclear cells in marginal periodontitis of patients with Down's syndrome. An indirect immunofluorescence technique on cryostat sections was used to study the cellular composition in chronic marginal periodontitis (CMP) of patients with Down's syndrome (DS). The findings were compared with CMP lesions in otherwise normal patients (NP). The distribution and amount of CD22+ cells (B lymphocytes), CD3+ cells (pan T lymphocytes), CD4+ cells (helper T subset), CD8+ cells (suppressor/cytotoxic T subset), and CD11c+ cells (in tissue, mainly monocytes and macrophages) were investigated. Morphologic studies showed a denser inflammatory infiltrate in DS than in NP. Countings showed significant differences in cell distribution (p = 0.0003) and cell profiles (p = 0.0273) between the two groups. The median CD4+/CD8+ ratio in DS (2.73) was significantly higher (p = 0.0024) than found in gingival inflammatory lesions from NP (1.08). The present study shows that DS patients have a different, more pronounced, immune response in CMP than NP.


The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary. The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate-limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post-natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By postnatal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post-natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post-natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 +/- 0.425 ng at PN 4, 86.31 +/- 20.42 ng at PN 7, 168.72 +/- 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 +/- 0.01% to 6.5 +/- 0.002% at PN 4, and continued to decrease until a level of 3 +/- 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6-hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 +/- 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 +/- 0.104 ng; PN 6 [DA] = 11.60 +/- 2.24 ng; PN 7 [DA] = 20.93 +/- 3.80 ng; and PN 10 [DA] = 27.95 +/- 3.46 ng. Melanotrope proliferation also increased in 6-hydroxydopamine-treated animals. At PN 4, the onset of innervation reduced the pre-innervation proliferation index to 8.75 +/- 0.002%, only a 30% reduction in contrast to the greater than 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6-OHDA administration. Our results demonstrated the time of dopamine innervation onset and a characteristic developmental pattern for axons within the rat intermediate lobe. The onset of innervation and increased dopamine concentration suggests increased dopaminergic control of the melanotropes, illustrated specifically by a decrease in their level of proliferation. This is the first presentation of evidence showing that dopaminergic innervation within the intermediate lobe of the rat pituitary regulates melanotrope proliferation.


Noradrenaline- and glutamate-induced taurine release from bulk isolated adult rat astrocytes. The influence of noradrenaline and various agonists of glutamatergic receptors on preloaded [3H]taurine release from bulk isolated adult rat brain astrocytes was investigated by a superfusion technique. In the presence of 1 mM noradrenaline a stimulation of taurine release, resembling that observed in astroglial cultures, was preceded by an inhibition of the efflux, thus demonstrating different dynamics of noradrenaline-evoked taurine release from that observed with beta-agonists on cultured astroglia. Application of 1 mM glutamate and kainate produced stimulation of the release, while 1 mM N-methyl-D-aspartate (NMDA) and 1 mM NMDA together with 65 mM K+ had no effect on the [3H]taurine release. These data suggest the presence of kainate-sensitive and the absence of NMDA-sensitive glutamate receptors on bulk isolated astrocytes, which is consistent with previous observations on astrocytes in culture.


Study of the mechanism of prostacyclin (PgI2) action on myocardial contractility. The experiments done on the isolated right ventricle of rat heart suspended in the bath for isolated organs with oxygenated Tyrode's solution showed the PgI2 (2.3 x 10-6 mmol) produced its positive inotropic effect indirectly by promoting both beta-adrenoreceptors and calcium entry through slow calcium channels because this effect could be blocked by propranolol (2.3 x 10-2 mmol - beta-adrenoreceptor blocker) or verapamil (4.3 x 10-3 mmol - slow calcium channel blocker).


Involvement of arachidonic acid and its metabolites in the inhibitory effect of beta-adrenoceptor blocking drugs on blood platelets. Propranolol (PRO), alprenolol (ALP), metipranolol (MET) and oxprenolol (OXP) inhibited arachidonic acid (AA) liberation from membrane phospholipids, malondialdehyde (MDA) formation and thromboxane B2 (TXB2) production in stimulated platelets. The inhibition was dose-dependent and with slight variations followed the rank order of potency: PRO greater than or equal to ALP greater than or equal to MET greater than or equal to OXP. Atenolol (ATE) was without any inhibitory effect. Inhibition of the arachidonic acid pathway in stimulated platelets may significantly contribute to the antiaggregatory effect of beta-adrenoceptor blocking drugs.


[Anterior urethral diverticular stones and retrocaval ureter in male: a case report and review of literature in Japan]. A case of diverticular stones in the male anterior urethra with retrocaval ureter is reported. A 26-year-old man visited our hospital for examination, who had experienced spontaneous stone discharge a few days earlier. Computed tomographic (CT) scan with ureteral catheterization and urethrography revealed a retrocaval ureter and urethral diverticular stones. Resection of urethral diverticulum with 7 stones and right ureteroplasty were performed. The urethrography and drip infusion pyelography (DIP) 9 months after operation showed no abnormal findings. The largest stone was 28 x 22 x 20 mm in size and 20 g in weight. The main components were ammonium dihydrogen-urate (70%), carbonate apatite and struvite. Histological feature of the epithelium of the urethral diverticulum indicated normal skin with hairs. Pathological diagnosis was para-urethral dermoid cyst. Our case is the 67th case of the male urethral diverticular stones and the first case of those with retrocaval ureter in Japan.


Pulmonary artery disease in Takayasu's arteritis: angiographic findings. One hundred sixteen arteriographic examinations in 98 patients with Takayasu's arteritis were studied retrospectively to evaluate the extent of aortic and pulmonary disease. Stenosis was the most frequent finding in the aorta and its branches, but occlusion, dilatation, and aneurysms were also seen. Adventitial vascular structures, consistent with dilated vasa vasorum, were observed in five patients (5%) and systemic artery-pulmonary artery communication was seen in six (6%). Twenty-one (70%) of 30 patients who had pulmonary arteriography were shown to have pulmonary artery involvement. Upper lobe pulmonary arterial branches showed abnormalities most frequently; segmental branches, followed by subsegmental branches, were most often involved. The frequency of abnormal findings on pulmonary arteriograms correlated with the number of involved brachiocephalic vessels. Stenotic lesions in the aorta and its branches progressed in five (45%) of 11 patients, and those in the pulmonary artery progressed in one (33%) of three cases. Takayasu's arteritis characteristically involves the systemic and pulmonary arteries, and the extent of arteritis in the major branches of the aortic arch appears to correlate with pulmonary arterial involvement. Both thoracic and abdominal aortographic studies and pulmonary arteriography are necessary to properly diagnose the extent of the disease, and angiography should be repeated for appropriate patient follow-up.


Predictors of the risk of development of acquired immunodeficiency syndrome within 24 months among gay men seropositive for human immunodeficiency virus type 1: a report from the Multicenter AIDS Cohort Study. The natural history of infection with human immunodeficiency virus type 1 (HIV-1) is characterized by a relentless decline in CD4-positive lymphocytes and the ultimate development of acquired immunodeficiency syndrome (AIDS). However, variables other than the CD4-positive lymphocyte level contribute to the measurement of risk for AIDS and can be used as predictors of AIDS onset. This study was undertaken to identify factors that, independently of the CD4-positive lymphocyte level, would predict the risk of AIDS over 24 months in a cohort of HIV-1 seropositive homosexual men receiving no antiretroviral therapy. Demographic, clinical, and laboratory data from 1,325 white, HIV-1 seropositive participants in the Multicenter AIDS Cohort Study who have been studied for 4 years were analyzed with univariate and multivariate methods. To control for stage of infection, the baseline percentage of CD4-positive lymphocytes (a known marker of disease progression), and the decline of CD4-positive cells during the first 6 months of observation were used as continuous variables. The variables that were independently associated with an increased risk of developing AIDS were: low baseline CD4 percentage, decline in the CD4 percentage during the first 6 months of follow-up, the presence of serum immunoglobulin A at baseline, decrease in hemoglobin during the first 6 months of follow-up, incident fatigue, and the interaction of decline in the CD4 percentage and incident thrush. While low CD4 percentage and other variables have been previously described as prognostic markers, decline in the CD4 percentage and the interaction of that decline and incident thrush have not previously been described as being of prognostic importance. These variables and the analytic method for estimating prognosis may prove useful for selecting and evaluating antiretroviral therapy, instituting prophylactic measures against certain opportunistic infections, and recruitment into clinical trials. Because study participants received no antiretroviral prophylaxis during the period under analysis, the method could be used to estimate the prognosis for those receiving investigational treatment were they to remain untreated, effectively making any participant in a clinical trial his own untreated control.


PCNA expression in cutaneous keratinous neoplasms and verruca vulgaris. Using an antibody to PCNA and a standard immunohistochemical system, the authors examined normal epidermis and cutaneous neoplasias for expression of PCNA, a protein associated with DNA polymerase delta and DNA replication. In squamous cell carcinoma in situ (SCCI), a unique expression of PCNA, which frequently involved the nuclei of all keratinocytes within the lesion, was found. Heaviest staining was in the uppermost layers of the epidermis. PCNA expression ended abruptly at the histologic margin of the lesion. Because SCCI can be associated with the presence of human papillomavirus (HPV) DNA, the authors evaluated PCNA expression in verruca vulgaris and found a pattern similar to that in SCCI. Assuming that PCNA expression in these two lesions is related to cell division, the authors hypothesize that the mechanisms that control proliferation in SCCI may be similar to those operative in verruca vulgaris.


Inconsistency of the immunophenotype of Reed-Sternberg cells in simultaneous and consecutive specimens from the same patients. A paraffin section evaluation in 56 patients. Both immunophenotypic overlaps between Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL), and evolution of one into the other have been reported. However, the underlying assumption that the antigenic expression of Reed-Sternberg (RS) cells is consistent in the same patient has not been evaluated. Such an evaluation was undertaken by immunophenotyping paraffin-embedded lymphoid tissue biopsies with HD from 56 patients in whom multiple specimens were obtained, either simultaneously from different sites or at different times. The panel of antibodies we used included: CD3 polyclonal antiserum, DAKO-M1 (CD15), L26 (CD20), BerH2 (CD30), MT1 (CD43), DAKO-LCA (CD45RB), UCHL1 (CD45R0), LN2 (CD74), and DAKO-EMA. The phenotype of RS cells was identical in simultaneous biopsies in only 11 of 39 patients (28%) and remained constant in consecutive biopsies in only 4 of 21 patients (19%). Major differences (relative to cell lineage specific antigens) were observed in 10 of 39 patients with simultaneous biopsies and in 10 of 21 patients over time; they mainly involved expression of T-cell antigens. Minor differences (relative to any other antigen) were observed in 22 of 39 patients with simultaneous biopsies and in 15 of 21 patients over time; these mainly involved CD15 or CD74. This striking variability of the immunophenotype of RS cells in the same patient may be due to aberrant marker expression, as a result of the neoplastic state, and/or to modulation of antigenic expression in relation to the host environment. This inconsistency suggests caution when interpreting the relationship between HD and NHL by paraffin immunophenotyping alone.


Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis. Capillary electrophoresis (CE) with a sieving buffer containing ethidium bromide was applied to the detection of PCR-amplified RFLP samples. With CE, in contrast to agarose gel electrophoresis, run times are short, i.e., typically less than 30 min, the capillary can be re-used, and full automation is feasible. The addition of ethidium bromide to the buffer system in conjunction with a field amplification injection technique led to increased sample detectability and resolution. Migration time precision was better than 0.2% RSD with a approximately 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Special software was used to correct for run-to-run migration time variations, thus facilitating genotype assignment.


Bauhinia purpurea--a new paraffin section marker for Reed-Sternberg cells of Hodgkin's disease. A comparison with Leu-M1 (CD15), LN2 (CD74), peanut agglutinin, and Ber-H2 (CD30). Thirty-three cases of Hodgkin's disease (thirteen nodular sclerosis, four diffuse, lymphocyte predominance, and sixteen mixed cellularity) were studied with Bauhinia purpurea (BPA), peanut agglutinin (PNA), anti-Leu-M1, LN2, and Ber-H2 by the avidinbiotin-peroxidase complex (ABC) method in paraffin sections. Reed-Sternberg (RS) cells and variants were stained positively with one or more of the reagents in all cases. BPA staining was positive in 32 of 33 cases (97.0%), PNA staining was positive in 23 of 33 cases (69.7%), Leu-M1 was positive in 13 of 33 cases (39.4%), LN2 was positive in 14 of 33 cases (42.4%), and Ber-H2 was positive in 24 of 33 cases (72.7%). Many RS cells were stained moderately to strongly and were readily recognized in 31 cases (96.9%) of BPA+, 10 (43.5%) of PNA+, 8 (61.5%) of Leu-M1+, 6 (42.9%) of LN2+, and 22 (91.7%) of Ber-H2+ cases; in the remaining positive cases, the RS cells were found only after careful searching. Three staining patterns were recognized: paranuclear, diffuse cytoplasmic, and membranous. These three patterns were obtained with all markers except for LN2. LN2 showed diffuse cytoplasmic staining in most of the positive cells, and a few cells showed paranuclear deposits. BPA reactivity was not affected by formalin fixation or paraffin embedding. Except for RS cells, BPA also showed dense cytoplasmic staining reaction with macrophage-histiocytes. Sixty cases of non-Hodgkin's diffuse lymphomas (30 T- and 30 B-cell origin) were also studied. Tumor cells were not stained with BPA, PNA, and Leu-M1, but stained positively with LN2 in six T-cell lymphomas and thirteen B-cell lymphomas, and with Ber-H2 in six T-cell lymphomas and one B-cell lymphoma. In conclusion, to facilitate the detection of RS cells and related variants in paraffin sections, BPA can be accepted as a useful marker due to its high-detection rate, reproducible staining pattern, and resistance to fixatives.


Assay of apical membrane enzymes based on fluorogenic substrates. A series of enzymatic rate assays are described. The assays are based on coumarin derivatives that are fluorogenic substrates for the enzymes dipeptidase IV, aminopeptidase N, alkaline phosphatase, and gamma-glutamyltransferase. These simple assays are rapid and offer improved sensitivity over established colorimetric methods. The substrates have apparent affinities for the enzymes of 5-250 microM. L-Glutamic acid gamma-(7-amido-4-methylcoumarin) is characterized as a substrate of gamma-glutamyltransferase on the basis of inhibition of enzymatic cleavage when the glycylglycine acceptor molecule is omitted and inhibition of the enzymatic reaction by addition of glycine. Assay conditions for the four enzymes are established such that less than 0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Intestinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays.


Determination of glutamic acid decarboxylase activity and inhibition by an H2O2-sensing glutamic acid oxidase biosensor. The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamate-selective biosensor. The biosensor is composed of an amperometric H2O2 electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H2O2 produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to alpha-ketoglutaric acid. Since GA is transformed to gamma-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental results and ease, time, and cost of operation.


A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity. A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.


Esmolol is more effective than sodium nitroprusside in reducing blood loss during orthognathic surgery. The goal of this study was to compare the efficacy of esmolol and sodium nitroprusside (SNP) as primary drugs for producing controlled hypotension and limiting blood loss during orthognathic surgery. Thirty ASA physical status I and II patients (mean age 22 yr) undergoing LeFort I maxillary osteotomies were randomly assigned to receive either esmolol (n = 15) or SNP (n = 15) as the primary drug to induce hypotension. All patients received a balanced anesthetic technique including isoflurane, with controlled hypotension during the downfracture of the maxilla. Patients assigned to the esmolol treatment group received boluses of 500 micrograms/kg of esmolol, followed by a continuous infusion of 100-300 micrograms.kg-1.min-1, and the SNP treatment group received a continuous infusion of SNP at 0.25-4.00 micrograms.kg-1.min-1; both infusions were titrated to obtain a mean arterial blood pressure within the target range of 55-65 mm Hg. The mean arterial blood pressure during the hypotensive period was 58.7 +/- 0.7 (mean +/- SEM) and 61.8 +/- 0.4 mm Hg for esmolol and SNP, respectively (P less than 0.001). In addition, 40% +/- 4% of the observed values in the esmolol group and 53% +/- 3% in the SNP group were outside the target range for mean arterial blood pressure (difference significant at P less than 0.05), and a greater proportion of the deviations were above 65 mm Hg in the SNP group than in the esmolol group (0.64 vs 0.46, respectively, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Intracranial pressure effects of dexmedetomidine in rabbits. The alpha 2-adrenergic receptor agonist dexmedetomidine produces an anesthetic state in a variety of species. Although its effects on cerebral blood flow and the electroencephalogram have been investigated, the effect of this drug on intracranial pressure (ICP) has not been reported previously. Dexmedetomidine therefore was intravenously administered to 24 New Zealand white rabbits that had been anesthetized with halothane and mechanically ventilated to maintain a constant arterial CO2 tension (PaCO2) between 34 and 39 mm Hg. After placement of an arterial catheter and ventricular cannula, baseline measurements of monitored variables, including heart rate, mean arterial blood pressure, ICP, end-tidal CO2, body temperature, and arterial blood gases, were recorded. Dexmedetomidine (20, 80, or 320 micrograms/kg IV) or saline solution was then infused over a 10-min period. The ICP transiently decreased by 31% in the 20-micrograms/kg group (from a mean value of 9.4 +/- 1.3 [SEM] to 6.5 +/- 1.0 mm Hg, P less than 0.05). In the 320-micrograms/kg group, ICP remained unchanged over the course of the study despite a significant increase in arterial blood pressure (32 mm Hg). The effects of dexmedetomidine on ICP were next investigated in the presence of intracranial hypertension produced by a cryogenic lesion (mean baseline ICP 16.8 mm Hg). In addition to the previously monitored variables, sagittal sinus blood flow was measured by the hydrogen clearance technique before and after the administration of dexmedetomidine (320 micrograms/kg IV). In these experiments, dexmedetomidine was associated with a 14% decrease in sagittal sinus blood flow that was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)


Alcohol self-administration: role of mesolimbic dopamine. It appears clear that ethanol reinforcement, like that of many abused drugs, utilizes the mesolimbic DA pathways. From the data presented on microinjection of DA agonists and antagonists, it would seem that only part of the regulatory process controlling ethanol drinking is directly involved with this pathway. Once drinking has begun, the DA antagonist raclopride results in a rapid termination of drinking. This appears to be a blocking effect of what may be conditioned reinforcement resulting from prior ethanol reinforcement initiation procedures. Microinjection of the DA agonists d-amphetamine and quinpirole prolonged drinking, with little signs of normal termination apparent in the 30-min session in many animals. This appeared to be the result of interference with normal termination processes. While it remains to be demonstrated that oral ethanol consumption results in the release of DA in the nucleus accumbens, evidence from prior work and the present studies support a role for the mesolimbic DA system in ethanol reinforcement.


Tooth mutilations in pre-Columbian Peru and Chile and modern-day Nigeria. Tooth mutilation or adornment in ancient Peru and Chile is discussed as well as that seen in present day Nigeria. Dental mutilation must be recognized (diagnosed) for what it is and discouraged in order to prevent dentoalveolar pathology and tooth loss.


Ethacrynic acid inhibition of microtubule assembly in vitro. Ethacrynic acid (ECA) is a sulfhydryl reactive diuretic drug. Recent studies show that ocular administration of ECA may have potential efficacy for treatment of glaucoma. ECA affects cell shape in cultured cells from the eye outflow pathway and the microtubule system is disrupted. We have studied the effect of ECA on microtubule protein (MTP) (tubulin and microtubule-associated proteins) and purified tubulin assembly. Fifty percent inhibition of MTP (1.8 mg/ml) assembly was found at 70 microM ECA in buffer and 410 microM ECA in 30% glycerol in buffer. If all sulfhydryl groups were attributed to tubulin, then approximately two sulfhydryls were blocked at 50% inhibition. Tubulin (2 mg/ml) assembly showed 50% inhibition at 175 microM ECA and approximately 2 sulfhydryl groups were lost. Increasing ECA preincubation times (0-60 min) with tubulin showed that the longer the preincubation time, the longer the lag time, and the slower the rate of assembly and that the percentage of inhibition was proportional to the ECA preincubation time. The number of blocked sulfhydryls also increased with preincubation time. Approximately two sulfhydryls were blocked at 50% inhibition of assembly. The critical concentration for assembly increased twofold when tubulin was preincubated with 0.1 mM ECA, suggesting a loss of active tubulin. Fifty percent inhibition of taxol-induced MTP and tubulin assembly occurred at 190 and 280 microM ECA, respectively, with 3.6 to 3.8 sulfhydryls blocked, respectively. Taxol protects microtubules from disassembly by ECA, suggesting that the ECA binding key sulfhydryls are blocked in the microtubule. These results suggest that ECA reacts slowly with tubulin and blocks sulfhydryl groups important for assembly. Microtubule-associated proteins and glycerol protect the sulfhydryls and so more ECA is necessary to inhibit assembly. Since the number of blocked sulfhydryls is greater at 50% inhibition for taxol-induced microtubules, sulfhydryl blocked tubulin incompetent to assemble under normal conditions may be induced to do so with taxol.


Characterization of a full-length, active-site mutant of diphtheria toxin. A full-length recombinant mutant of diphtheria toxin containing serine in place of a crucial active-site glutamate has been purified and characterized. The serine substitution caused a minor structural alteration in the toxin as measured by trypsinolysis. ADP-ribosyltransferase activity and cytotoxicity of the mutant were both decreased by approximately 500-fold. A similar reduction in cytotoxicity was found when the enzymic fragments of both the wild-type and mutant toxins were introduced into the cytosol of fibroblasts by osmotically lysing pinosomes. The mutation did not alter the binding of the toxin to cell surface receptors and had no apparent effect on membrane translocation. The results suggest that the decreased cytotoxicity of the mutant is solely due to the reduced ADP-ribosyltransferase activity.


Ankylosing spondylitis and HLA-B27: restriction fragment length polymorphism and sequencing of an HLA-B27 allele from a patient with ankylosing spondylitis. Two groups of patients with ankylosing spondylitis (AS) from England and Poland were examined for restriction fragment length polymorphisms (RFLPs) associated with the disease. No preferential association was found between the 9.2 kb PvuII fragment in HLA-B27 positive patients with AS compared with HLA-B27 healthy subjects as had been previously reported. In the English group, however, a 14 kb PvuII fragment was more common in HLA-B27 positive subjects with AS than in normal controls. Also 4.6 and 3.7 kb PvuII fragments were more prevalent in subjects without AS than in the group with AS, but these results were confined to the English group. Furthermore, the sequence of an HLA-B*2705 gene isolated from a patient with AS was examined, and no significant differences were found compared with the sequence isolated from a healthy subject. There do not seem to be significant genetic differences in the coding or in the regulatory region in HLA-B27 alleles, in subjects with or without AS.


Induction of remission in a patient with Takayasu's arteritis by low dose pulses of methotrexate. A 42 year old woman with Takayasu's arteritis responded to treatment with prednisone (60 mg daily) but developed severe side effects. Cyclophosphamide treatment did not produce a clinical improvement or steroid sparing effect. A treatment trial with a weekly pulse of low dose methotrexate improved her symptoms and the patency of her occluded left subclavian artery.


The Zollinger-Ellison syndrome. A collective surgical experience. A retrospective study of 90 surgically treated patients with the Zollinger-Ellison syndrome seen from 1958 through 1990 was performed. Fifteen patients had Zollinger-Ellison syndrome as a manifestation of multiple endocrine neoplasia type I. Preoperative tumor localization was positive in 46% of 54 patients studied. Gastrinomas were identified in 66% of patients, 38% of the tumors being malignant. Postoperative eugastrinemia was achieved in 11% of patients after a variety of surgical procedures. Exploratory laparotomy provides the only chance for cure and identifies the significant prognostic factors associated with long-term patient survival: small tumor size, extrapancreatic primary, and absence of tumor metastases.


Correlation among oxysterol potencies in the regulation of the degradation of 3-hydroxy-3-methylglutaryl CoA reductase, the repression of 3-hydroxy-3-methylglutaryl CoA synthase and affinities for the oxysterol receptor. 25-Hydroxycholesterol regulates cholesterol biosynthesis by two mechanisms: repression of the transcription of the genes for several cholesterogenic enzymes and acceleration of the degradation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase. In the present work the structural features which govern oxysterol potency were determined separately for each regulatory mechanism. Regulation of degradation was tested using a 3-hydroxy-3-methylglutaryl CoA reductase-beta-galactosidase fusion protein. Repression of enzyme synthesis was tested by measuring 3-hydroxy-3-methylglutaryl CoA synthase activity since this protein is not regulated by a degradative mechanism. Oxysterol activities were highly correlated between the two assays (R = .959) demonstrating that the degradative and repressor mechanisms share an element which determines oxysterol regulatory potency. Correlation of these results with previous data for the affinity of these oxysterols for the oxysterol receptor suggests that the receptor is the element involved in both these regulatory pathways.


Inhibition of receptor-stimulated guanylyl cyclase by intracellular calcium ions in Dictyostelium cells. In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P3 which releases Ca2+ from internal stores. The following data indicate that intracellular Ca2+ ions inhibit guanylyl cyclase activity. 1) In vitro, Ca2+ inhibits guanylyl cyclase with IC50 = 41 nM Ca2+ and Hill-coefficient of 2.1. 2) Extracellular Ca2+ does not affect basal cGMP levels of intact cells. In electro-permeabilized cells, however, cGMP levels are reduced by 85% within 45 s after addition of 10(-6) M Ca2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca2+. 3) Receptor-stimulated activation of guanylyl cyclase in electro-permeabilized cells is also inhibited by extracellular Ca2+ with half-maximal effect at 200 nM Ca2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase.


Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group. The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.


Differential expression of the neu oncogene in mouse liver and pancreatic cell lines. To study the tissue-specific expression of neu oncogene, we used two mouse tumor cell lines derived from liver, Hep1-a, and pancreatic, 266-6, tumors as a model system. The endogenous neu gene is expressed in Hep1-a but not in 266-6 cells. We demonstrate in this report that differential expression of the neu gene in these two cell lines is mainly regulated at transcriptional level. The neu promoter sequence responsible for the differential regulation is localized within a 90 bp region and it is possibly due to lack of a specific positive transcription factor(s) interacting with this region in 266-6 cells.


Hsp60/chaperonin gene expression and differentiation of human colon adenocarcinoma and multipotent leukaemic cells. Elevated mitochondrial gene expression is an early event in the switch from proliferation to differentiation of the human colon adenocarcinoma cell line, HT29, promoted by trehalose replacement of exogenous glucose. Here we report the isolation and elevated expression of hsp60, the gene encoding chaperonin, a mitochondrial protein required for assembly of mitochondrial and cellular proteins. In contrast to HT29, leukaemic cells (HL60 and K562) neither differentiated nor altered their mitochondrial gene expression after treatment with trehalose. However, differentiation of these cells, as promoted by 12-O-tetradecanoylphorbol-13-acetate actually resulted in decreased levels of hsp60 mRNA expression as well as mitochondrial RNA expression, suggesting significant differences in involvement of mitochondria in the differentiation of these cell lineages.


An immunomodulatory protein, Ling Zhi-8, facilitates cellular interaction through modulation of adhesion molecules. Ling Zhi-8 (LZ-8), a novel immunomodulatory protein, markedly enhanced the expression of CD11b, but not CD11a, CD13, CD14, CD18, CD33 or HLA-DR, on the U937 cell line in a dose-dependent fashion. It also induced ICAM-1 expression on vascular endothelial cells and significantly augmented gamma - interferon-induced cellular binding between vascular endothelial cells and U937. Furthermore, LZ-8 increased the expression of CD2, but not VLA4, VLA5 or LFA3, on MOLT4 and enhanced rosette formation between human T cells and sheep red blood cells. These data suggest that LZ-8 exerts its pharmacological effect by modulating adhesion molecules on immunocompetent cells.


Is a point mutation in the mitochondrial ND2 gene associated with Alzheimer's disease. A specific mitochondrial DNA mutation at position 5460 in the ND2 gene of the human mitochondrial genome was recently reported to exist in 10 of 19 patients with Alzheimer's disease, implying an association between this mtDNA mutation and the occurrence of the disease. We have analyzed tissues from 15 patients with Alzheimer's disease for the presence of the ND2 mutation, and have not been able to confirm these findings. We believe that this mutation is not specifically associated with Alzheimer's disease, but rather, is a neutral polymorphism present in the population.


Stearoyl-CoA desaturase gene expression in lymphocytes. B lymphocytes from the spleens of normal (BALB/c) and autoimmune (MRL/lpr) strains of mice express the SCD-2 form of stearoyl-CoA desaturase as opposed to the SCD-1 form of the gene which is expressed in liver. However, whereas BALB/c T cells did not express SCD-1 or SCD-2, both BALB/c thymocytes and MRL/lpr T cells expressed SCD-2, suggesting a developmental down-regulation of SCD-2 within the T cell lineage. Northern analyses also revealed the expression of SCD-2 in the T cell lines BW5147, CTLL-2 and HT-2 and in BCL1, a B cell line. SCD-1 expression was not detected in any of the lymphoid cells tested. Finally, we show that SCD-2 gene expression is inhibited by arachidonic acid (20:4). These results demonstrate the complexity of SCD-2 regulation in lymphoid cells.


Stereoisomers of calcium antagonists which differ markedly in their potencies as calcium blockers are equally effective in modulating drug transport by P-glycoprotein. The (-)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer. However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs [Gruber et al., Int J Cancer 41: 224-226, 1988]. In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyridine structures which markedly differ in their potency as calcium antagonists. We have tested these drugs for their ability to increase intracellular accumulation of [3H]vinblastine ([3H]VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells. Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis. The enantiomers with phenylalkylamine structures [(+/-)-verapamil; (+/-)-devapamil; (+/-)-emopamil)] and with dihydropyridine structures [(+/-)-isradipine; (+/-)-nimodipine; (+/-)-felodipine; (+/-)-nitrendipine; (+/-)-niguldipine] increased [3H]VBL accumulation in both cell lines at micromolar concentrations. Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells. There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular [3H]VBL transport. Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs. Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers. Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another useful candidate drug for the treatment of multidrug resistance in cancer patients.


Apolipoprotein B gene polymorphisms, lipoproteins and coronary atherosclerosis: a study of young myocardial infarction survivors and healthy population-based individuals. Association studies were carried out in a sample of 87 patients from Sweden who had survived a myocardial infarction (MI) before the age of 45, and 91 age-matched healthy individuals, to compare the impact of polymorphisms at the apolipoprotein (apo) E and B gene loci on among-individual differences in plasma lipid traits and progression of atherosclerosis. In the group of healthy individuals, polymorphisms creating the common apo E isoforms were, as expected, associated with significant differences in total and low density lipoprotein (LDL) cholesterol (11.7% and 11.6% of sample variance). For apo B, the polymorphism with the largest effect on apo B levels (16% of sample variance) was the C to T transition 265 bp 5' of the cap site, in the promoter (detectable by MspI). Both this polymorphism and the threonine2488 neutral substitution (detectable by XbaI) were associated with significant effects on LDL-cholesterol (8.3% and 9.3% of sample variance, respectively). The asparagine/serine4311 polymorphism was associated with a significant effect on high density lipoprotein (HDL) cholesterol alone, and there was no significant association with the glutamate/lysine4154 polymorphism (detectable by EcoRI) or the leucine-alanine-leucine (LAL) insertion/deletion polymorphism in the signal peptide. In the patients, polymorphisms creating the three common apo E isoforms were associated with large effects on cholesterol, apo B and triglyceride levels (19.9%, 20.3% and 23.9% of sample variance) of similar magnitude as in the healthy individuals. Apo B polymorphisms were found to be associated with much smaller effects on lipid traits than in the healthy individuals. The only significant association was between the asparagine/serine4311 polymorphism and HDL-triglyceride levels. However, global severity of coronary atherosclerosis at the first angiography was found to be significantly associated with the LAL insertion/deletion polymorphism (P = 0.008). Thus variation at the apo B gene locus is associated with the development of atherosclerosis, but the data suggests that this may act through mechanisms not directly related to effects on measured lipid traits.


The MspI restriction fragment length polymorphism 3' to the apolipoprotein A-II gene: relationships with lipids, apolipoproteins, and premature coronary artery disease. In previous studies, a restriction fragment length polymorphism (RFLP) has been identified using MspI restriction endonuclease in the 3' region of the apo A-II gene. The rare variant site for this MspI (M2) has been reported to be associated with higher levels of HDL cholesterol and apo A-II. We have studied the frequency and lipid associations of this RFLP in a population of 168 coronary artery disease (CAD) male and female patients, who had more than 50% narrowing of one or more arteries prior to age 60 years, as well as 255 aged-matched males and females from the Framingham Offspring Study. We also studied 31 kindreds in which the proband had premature CAD. The frequency of the M2 allele was higher in CAD cases (0.20) than in the controls (0.13) (P less than 0.05). In general, those subjects carrying the M2 allele had lower HDL cholesterol and apo A-I plasma levels; however, this difference was only significant (P less than 0.02 and 0.002, respectively) in females with CAD. No cosegregation of the M2 allele with hypoalphalipoproteinemia was found in 31 kindreds studied. However, in both generations there was a trend for those subjects carrying the M2 allele to have lower HDL cholesterol levels than those carrying the M1 allele. Sequence analysis of the apo A-II gene of subjects homozygous for either the M1 (n = 1) or the M2 allele (n = 2) revealed that this RFLP is due to a T----C single base mutation 528 bp 3' to the apo A-II gene. In the subjects homozygous for the M2 allele no other mutations were found within the coding region of the apo A-II gene that could result in changes in the primary sequence of the protein. These data indicate that the MspI RFLP 3' to the apo A-II gene is somewhat more frequent in the CAD group. However, there was no significant association between this RFLP and any of the parameters examined. In conclusion, this DNA marker lacks the specificity to be clinically useful for CAD risk assessment in the population studied.


High density lipoproteins: physiopathology and clinical relevance. On September 13-14 1991 a Symposium on High Density Lipoproteins: Physiopathology and Clinical Relevance was held in Bellagio (Italy). This Symposium was aimed at discussing various aspects of HDL from epidemiology to the most recent advances in the understanding of HDL metabolism and factors (diets, drugs) affecting their levels.


The acute effects of a loading dose of phenylalanine in unipolar depressed patients with and without tardive dyskinesia. The effects of a loading dose of 100 mg/kg phenylalanine (PHE) were assessed in three groups of DSM-III-R diagnosed unipolar depressed patients: patients with tardive dyskinesia (TD) (n = 11); patients exposed to neuroleptics (NLs) but without TD (n = 10); and patients never exposed to NLs (n = 10). No significant differences were obtained in fasting and 2 hour postloading PHE plasma levels between the groups. A statistically significant correlation was found between Abnormal Involuntary Movements Scale total scores and postloading PHE plasma levels (p less than .05). Three TD patients showed unusually large increases in PHE plasma levels and PHE:large neutral amino acid ratios. Abnormalities in PHE metabolism may contribute to the development and severity of TD in some NL-treated unipolar depressed patients.


Management of essential hypertension in patients with different degrees of left ventricular hypertrophy. Multicenter trial. Three hundred and four hypertensive patients with different degrees of left ventricular hypertrophy (LVH) were recruited and followed for 4 years. The patients were randomized into two groups: Group I (150 patients) was treated with a combination of hypotensive drugs including beta-blockers, and group II (154 patients) was treated with the same combination of drugs including diuretics instead of beta-blockers. By the end of the fourth year, 60 endpoints were recorded: 17 strokes, 13 myocardial infarctions, and 30 cases of chronic coronary insufficiency. Mortality was statistically higher in group II (7 of 154 or 5% v 1 of 150 or 1%) (P less than .035), but there was no difference between the groups in the incidence of nonfatal endpoints. These data confirm that beta-blockers can reduce mortality associated with the complications presented in hypertensive patients. Increased left ventricular myocardial mass (greater than 200 g, according to Teichholz' formula) was shown to have prognostic value for the development of complications. In patients with LVMM greater than 200 g, the probability of fatal complications was higher (P less than .007), as was the probability of nonfatal myocardial infarction (P less than .01), development of coronary artery disease (P less than .02), and all complications (P less than .0003). Regression of LVH to less than 200 g (according to Teichholz' formula) improved prognosis.


B-HT 920 activates dopamine D2 receptors coupled to inhibition of adenylate cyclase activity. In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.


Monitoring glutamine in mammalian cell cultures using an amperometric biosensor. An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.