Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex. Reversal of the endogenous feedback inhibition by phosphorylation of serine-40.

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an S(eo),w value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & Haavik (1988) J. Biol. Chem. 263, 18621-18626]. In the rat PC12 enzyme, all of the iron present (0.53 +/- 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 +/- 0.05 mol of dopamine and 0.10 +/- 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of L-tyrosine a new rhombic (magnitude of E/D = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.


Differences in 2-oxoglutarate dehydrogenase regulation in liver and kidney.

In response to acidosis, renal ammoniagenesis is stimulated, enhancing urinary buffering power, while hepatic ammoniagenesis and ureagenesis decrease, so as to spare bicarbonate consumed in the urea cycle. 2-Oxoglutarate (2-OG) levels can regulate ammoniagenesis in kidney and gluconeogenesis in liver and kidney. Since the activity of 2-oxoglutarate dehydrogenase (2-OGDH) has an important influence on cellular levels of 2-OG, this study evaluated the effects of pH on 2-OGDH in liver and kidney and found that: (1) the isolated enzyme from both organs has the same pH-sensitivity; (2) 2-OGDH flux measured in intact mitochondria was inhibited by increasing H+ in liver, but stimulated in kidney; (3) transport of 2-OG into the mitochondria was not rate-limiting; (4) liver mitochondrial 2-OGDH exhibited a strong preference for 2-OG generated within the mitochondria from glutamate-oxaloacetate transaminase (GOT), suggesting that channelling between GOT and 2-OGDH occurs. Since complexation between 2-OGDH and GOT occurs in vitro, we propose that the degree of complexation is higher in liver than in kidney, such that most of the 2-OGDH may be complexed to GOT in liver. In the liver the inherent H(+)-sensitivity of 2-OGDH is masked by the pH-sensitivity of GOT and the glutamate-aspartate carrier.


G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.


HLA class II sequence polymorphisms and susceptibility to rheumatoid arthritis in Greeks. The HLA-DR beta shared-epitope hypothesis accounts for the disease in only a minority of Greek patients.

In Northern Europeans, rheumatoid arthritis (RA) is strongly associated with a relatively conserved pentapeptide sequence of HLA-DR beta found notably in the HLA-DR4 subtypes Dw4 and Dw14 and in DR1. A previous serologic study of HLA class II polymorphism in a Greek population with RA failed to show significant associations with any antigen. We characterized HLA-DRB polymorphisms in Greek patients with RA and in control subjects by restriction fragment length polymorphism analysis. Allelic DRB subtypes were examined by polymerase chain reaction amplification and oligonucleotide hybridization. DNA analysis in the RA patients showed that although individual HLA-DR allelic associations were weak, a relatively conserved HLA-DR beta motif was significantly associated with RA in this population of Greek patients. The third hypervariable region amino acid sequences QRRAA, QKRAA, or RRRAA were found in the HLA-DR beta 1 of 43.5% of the RA patients versus 15.5% of the controls (uncorrected P = 0.00004). Sequences shown to influence susceptibility to RA in patients in the UK also play a role in patients in Greece. However, 57% of Greek patients lack the putative HLA-DR beta motif, which suggests that considerable immunogenetic heterogeneity underlies disease susceptibility in this population.


T cell receptor gamma variable gene locus polymorphism in patients with rheumatoid arthritis.

We sought new susceptibility markers for rheumatoid arthritis (RA) among the T cell receptor gamma (TCR gamma) genes. We analyzed restriction fragment length polymorphisms (RFLP) of the first variable subgroup of TCR gamma genes in a group of French control subjects and a group of French RA patients. No significant difference in Eco RI RFLP was found between the 2 study populations: Allele frequencies were virtually identical. There was no polymorphism using Hind III. These results exclude TCRV gamma I polymorphism as a disease susceptibility marker in RA.


Follicular dendritic cells and their role in HIV infection.

Recently, much attention has focussed on the role of follicular dendritic cells as a reservoir of infectious particles in retroviral, particularly HIV, infection. In this report from a recent meeting, Johannes Gerdes and Hans-Dieter Flad describe these studies in the context of a growing awareness of the morphological, phenotypic and functional heterogeneity of the cells.


Motor activity and the mesotelencephalic dopamine function. I. High-resolution temporal and genetic analysis of open-field behavior.

In a genetic selection experiment whose goal was to construct congenic neurological animal model lines with different mesotelencephalic dopamine systems, we produced foundation F2 generations derived from crosses (C57BL/6ByJXBALB/cJ and C57BL/6ByJXCXBI/ByJ) between highly inbred mouse lines with different dopamine systems. In this report, temporal distribution, latency, and genetic variability of open-field (OF) behavioral variables were investigated in order to establish a behavioral profile for the various generations and to provide behavioral data as a first step towards multivariate studies on correlations between OF behaviors and mesencephalic and striatal tyrosine hydroxylase activity. Analysis of the behavioral data provided evidence that measures of the open-field behaviors varied significantly across time segments of the test and that the temporal profiles of several behavioral variables were genotype-dependent. It is suggested that (1) OF behaviors have dynamic temporal profiles, and (2) temporal-genetic analysis can be a useful auxiliary method in the functional interpretation of behavior.


A chimeric EGFR/neu receptor in functional analysis of the neu oncoprotein.

As the factor binding to the neu protein has been unknown, it has not been possible to confirm experimentally the proposed growth-factor receptor like functions of the neu protein. To approach this problem we constructed a recombinant receptor which enabled ligand regulation of the neu tyrosine kinase. The hybrid receptor consisted of the extracellular ligand binding, transmembrane and protein kinase C-substrate domains joined to the intracellular tyrosine kinase and carboxyl-terminal domains of the neu protein. Several properties of NIH3T3 cells carrying this construct were tested. We obtained the first experimental evidence that the neu proto-oncogene has mitogenic and transforming activities only in the presence of a ligand stimulating its tyrosine kinase activity. Various cellular and molecular biological parameters indicated that the chimeric receptor behaved very similarly to the EGFR. Also, this chimeric receptor has allowed us to compare the constitutive oncogenic and the ligand-activated non-oncogenic activities of the neu tyrosine kinase. In the future we plan to focus on characterization of possible differences between EGFR and neu signalling in more differentiated cellular backgrounds.


Mechanisms of multidrug resistance in cancer treatment.

Advanced breast cancer responds to a range of cytotoxic agents, but resistance always develops. Understanding the mechanisms of resistance may provide new therapeutic options. There are several major groups of resistance mechanisms. 1) The multidrug resistant phenotype. This is due to a membrane pump that can extrude a wide range of anticancer drugs--the P-glycoprotein. It is inhibited by a range of clinically used calcium channel blockers such as nifedipine and verapamil. Several other membrane proteins of 180 KD, 170 KD, 300 KD and 85 KD have been reported and are associated with MDR. 2) Glutathione transferences and detoxification mechanisms. These are a multigene family of enzymes that conjugate glutathione to chemically reactive groups. There are 3 major groups of enzymes--acidic, basic and neutral. They have been implicated in resistance to doxorubicin, melphalan cisplatinum chlorambucil and other alkylating agents. Other protecting systems include metallothionein and selenium dependent glutathione peroxidase. HSP27 confers doxorubicin resistance. 3) Topoisomerase II. DNA topoisomerases are involved in several aspects of DNA metabolism in particular genetic recombination, DNA transcription, chromosome segregation. They are a target for doxorubicin, mitoxantrone, VP16. Low levels of expression are associated with resistance. However, it is oestrogen inducible and this may be of therapeutic value. A novel topo IIb which is more drug resistant has been reported. 4) DNA repair. A score or more of genes are involved in the repair of DNA damage by drugs and radiation. Defective DNA repair may predispose to cancer of the breast and be responsible for adverse radiation reactions. Enhanced repair has been shown to be a mechanism of cisplatinum resistance. Several genes are inducible by DNA damage and may confer resistance e.g. A45. 5) Drug activation. Mitomycin C as well as cyclophosphamide and VP16 require activation for their effects. Low levels of cytochrome p450 reductase are associated with MMC resistance.


Multiple endocrine neoplasia type 2 with malignant pheochromocytoma--long term follow-up of a case by 131I-meta-iodobenzylguanidine scintigraphy.

The case of a 33-year-old Japanese man, who has Multiple Endocrine Neoplasia Type 2 (MEN IIa) (Sipple's syndrome) with malignant pheochromocytoma, is reported. He had survived for twelve years since the initial diagnosis of malignant pheochromocytoma. Within this period, he had undergone 131I-meta-iodobenzylguanidine scintigraphy twice, in 1983 and 1990. This is the first case in Japan of a longterm surviving patient with malignant pheochromocytoma followed up by 131I-MIBG scintigraphy. Although he had no exacerbation of clinical symptoms or urinary catecholamine levels, second scintigraphy clearly showed an increase in the tumor size, new metastasis of the malignant pheochromocytoma and exacerbation of the medullary thyroid carcinoma. Compared with any other roentgenological device and hormonal data, 131I-MIBG scintigraphy was seen to be a good tool for evaluating the localization and the progression of tumors. 131I-MIBG scintigraphy is a useful procedure not only for initial diagnosis but also for judging progression in a case of advanced malignant pheochromocytoma.


Intracranial germ cell tumour in association with cryptorchidism.

An intracranial germ cell tumour (GCT) in a patient with cryptorchidism is presented. The known association between testicular GCTs and cryptorchidism suggests a possible common predisposition for gonadal and extragonadal tumours. To confirm this the frequency of the reported association needs to be defined.


Response of V beta 8.1+ T cell clones to self Mls-1a: implications for the origin of autoreactive T cells.

Clonal deletion and anergy are two major mechanisms of self-tolerance. However, the molecular mechanisms underlying clonal deletion and anergy, as well as the threshold of TCR affinity/avidity required for these processes, are not known. Expression of the V beta 8.1 TCR correlates with the reactivity of the T cells to the minor lymphocyte stimulating locus-1a (Mls-1a) and T cells expressing this TCR are deleted in the thymus of Mls-1a mice. Similarly, in TCR V beta 8.1 transgenic mice, the number of CD4+CD8-T cells is reduced in Mls-1a mice. However, small numbers of CD4+CD8-T cells remain in the periphery of adult Mls-1a transgenic mice. We have generated T cell clones from TCR V beta 8.1 transgenic mice by stimulation of lymph node T cells with C57BL/6 alloantigens. Interestingly, CD4+CD8-V beta 8.1+ clones isolated from the transgenic mice of Mls-1a background responded to the self-antigen Mls-1a, to which they did not respond in primary assay. Reactive patterns of the clones were compared with clones derived from Mls-1b mice. Proliferation and cytokine production of the clones from Mls-1a mice to the self-antigen Mls-1a were generally reduced when compared with clones from Mls-1b mice. More importantly, T cell clones from Mls-1a mice required more Mls-1a antigen for their activation, and were more susceptible to the inhibitory effects of anti-CD4 antibody on the proliferative responses to Mls-1a than those from Mls-1b mice. These results suggest that the T cell receptor on clones derived from Mls-1a mice have functional but reduced affinity/avidity for self-antigen Mls-1a.


Influence of HLA-DR2, HLA-DPw4, and T cell receptor alpha chain genes on the susceptibility to multiple sclerosis.

In view of our recent report that T cell receptor (TCR) alpha chain restriction fragment length polymorphism (RFLP) is associated with multiple sclerosis (MS), we have assessed the possibility that HLA-DR2, HLA-DPw4, and TCR alpha chain RFLPs may interact to increase the risk of developing MS. Detection of TCR alpha chain polymorphisms, and HLA-DR and -DP typing were carried out by RFLP analysis on MS patients and healthy controls from the Melbourne metropolitan area. Interaction effects among these loci in producing susceptibility to MS were assessed by hierarchial log-linear analysis. Although HLA-DR2 was significantly associated with MS (chi 2 = 9.30, P = 0.002), no interactive effect between MS and HLA-DPw4 was observed. Significant interactions were observed with MS and both C alpha and V alpha, with the strongest effect seen with C alpha (chi 2 = 21.30, P less than 0.001). The combination of DR2/C alpha imparted a relative risk of 47. However, when the data were analysed for four and three way interactions, no significant effects were seen with MS, DR2, DPw4, V alpha, and C alpha, implying that the combined presence of these polymorphic markers is not essential for increasing susceptibility to MS.


Antigen-independent adhesion of CD45RA (naive) and CD45RO (memory) CD4 T cells to B cells.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)


cAMP-dependent protein kinase activation mediated by beta 3-adrenergic receptors parallels lipolysis in rat adipocytes.

Catecholamine-induced lipolysis is chiefly mediated through the recently characterized beta 3-adrenergic receptor (AR) in rat adipocytes. Discrepancies between the ability of beta 3-AR agonists to stimulate adenylyl cyclase and the resulting lipolysis were recently reported. cAMP-dependent protein kinase (A-kinase) activation induced by these agonists was compared to lipolysis. Agonist potencies were similar for A-kinase activity ratios and lipolysis. The same A-kinase activity ratio to lipolysis relationship was found for the beta 3-AR agonists tested.


Chlorophyll a/b-binding protein genes are differentially expressed during soybean development.

The levels of chlorophyll a/b-binding protein (Cab) gene polysomal poly(A)+ mRNA were quantitated throughout the development of Glycine max L. Cab mRNAs were abundant in young expanding leaves, representing 6.1% of the leaf mRNA population. Lower Cab mRNA levels were present in embryos, stems, and cotyledons of developing seedlings; the lowest levels were found in roots where they accounted for 0.04% of the polysomal poly(A)+ mRNA of this organ. To determine the contribution of different members of the Cab gene family to the Cab mRNA populations, a quantitative S1 nuclease reconstruction assay was developed. Cab3, Cab4, and Cab5 mRNAs were detected in all stages examined during soybean development but their levels underwent differential changes. Cab3 encodes the most abundant Cab mRNA in young leaves, developing embryos, and in Stage VII cotyledons from the developing soybean seedling. The levels of Cab mRNAs were compared to the levels of ribulose-1,5-bisphosphate carboxylase small subunit gene mRNA and differences in their patterns of accumulation were noted. Collectively these data indicate that during soybean embryogenesis developmental control mechanisms supersede light-regulatory signals.


Angiopeptin: a treatment for accelerated myointimal hyperplasia?

Extensive smooth muscle cell proliferation is the major event leading to the narrowing or occlusion of the lumen of the coronary arteries of patients undergoing heart transplantation or percutaneous transluminal coronary angioplasty. The smooth muscle cell proliferation in transplant atherosclerosis may be initiated by immunologic events, allowing the vascular smooth muscle cells to respond with migration and proliferation to circulating growth factors as well as chemoattractants and growth factors released by inflammatory cells and smooth muscle cells themselves. The somatostatin analog angiopeptin is a cyclic octapeptide that has different binding affinities for different somatostatin receptors compared with the parent compound itself. The antiproliferative effects of angiopeptin have been demonstrated in vitro in several tumor cell lines. In a rabbit model of heterotopic heart transplant-accelerated coronary atherosclerosis, angiopeptin has been shown to attenuate myointimal hyperplasia. Further studies in simpler models of myointimal hyperplasia have shown that angiopeptin will inhibit smooth muscle cell proliferation after vascular injury. Further, angiopeptin inhibits thymidine uptake in vitro in pig coronary arteries. Angiopeptin also exerts its inhibitory effect at a very early stage after injury: an 8-hour delay of treatment abolishes the inhibitory effect of angiopeptin on smooth muscle cell proliferation (intimal hyperplasia). On the basis of the experimental data, clinical studies of the inhibitory effect of angiopeptin on prevention of transplant atherosclerosis in heart transplant patients and prevention of restenosis after coronary artery angioplasty are ongoing, as well as are studies in patients undergoing saphenous vein coronary artery bypass surgery.


Neural transplants: prospects for Alzheimer's disease.

Neural transplantation has emerged as a useful tool for neurobiologists to investigate basic mechanisms of brain function, as well as potential therapies for neurodegenerative diseases. At present, intracerebral grafting is not a therapy for Alzheimer's disease (AD) or dementia. However, recent developments using molecular biological methodologies in combination with grafting have opened new avenues to explore new therapeutic approaches and address questions of etiology.


Basic mechanisms of the epilepsies.

To understand the fundamental pathophysiology of the epilepsies, the chronic changes of structure and function in the brain that distinguish them must be identified and the basic mechanisms by which these changes come about need to be delineated. Recently, progress has been made along these lines, identifying morphological and operational differences in epileptic brains, both those from humans and those from animals, as well as alterations in gene activation brought about by seizures.


The psychopharmacology of social phobia.

The use of psychotropic drugs in the treatment of social phobia has occurred only recently. The author reviews the available studies which suggest that medications in the beta-blocker group, the monoamine oxidase inhibitors, the benzodiazepines, and possibly others are useful in the treatment of social phobia.


Evaluation of abnormal biomechanics of the foot and ankle in athletes.

Athletes often suffer from recurrent or chronic overuse symptoms of the lower extremities. During the office visit it is essential to analyse the patient's shoes, gait cycle, lower extremities and, especially, the talocrural, subtalar and more distal joints of the ankle and foot. The basic (clinical) biomechanical analysis can be supplemented by radiographs, treadmill and video analysis and mirror table (podoscope) examinations. Ideally, successful pain relief by correction of the observed abnormality with an orthotic device completes the diagnostic procedure, especially if symptoms return soon after the removal of the device. In treatment custom-made, expensive orthotics should not be prescribed for overuse symptoms without an obvious malalignment, for asymptomatic athletes with a malalignment, or for symptoms in which the causal relationship between the biomechanical abnormality and symptoms is difficult to see. Strict indications for prescription of orthotics and close cooperation between the attending physician, physical therapist and orthotist are prerequisites for obtaining good, long-lasting results.


Backbone modifications in somatostatin analogues: relation between conformation and activity.

Twenty cyclic and linear analogues of somatostatin have been compared with respect to their conformational behavior and biological activity. It appears that all active compounds have in common a well defined, predominant backbone conformation. For linear peptides, this conformation can only be detected at low (-80 degrees C) temperature by NMR measurements. Selectivity is suggested to be determined by the nature and topology of the side chains linked to this common backbone conformation. The side chain conformation is also only accessible for NOE measurements in the low temperature range.


The versatile superficial inferior epigastric artery free flap.

A series of 27 successful Superficial Inferior Epigastric Artery free flaps were analysed for a number of clinical and surgical variables. The anatomy and surgical technique of the flap are detailed and its advantages and disadvantages relative to other free flaps used for soft tissue contour are considered. Analysis of the variety of recipient sites of this flap is presented, as are details of the vascular pedicle and our experience with its constancy. It has been found to be a safe flap in experienced hands, with an aesthetic donor site scar, no functional loss and it lends itself to subsequent recontouring with suction assisted lipectomy or open techniques.


The effects of pituitary stalk transection, hypophysectomy and thyroid hormone status on insulin-like growth factor 2-, growth hormone releasing hormone-, and somatostatin mRNA prevalence in rat brain.

We have used in situ hybridization histochemistry to determine the effects of pituitary stalk transection, hypophysectomy and drug-induced changes in thyroid status on mRNA levels encoding insulin-like growth factor 2, somatostatin, and growth hormone-releasing factor in the choroid plexus, hypothalamic periventricular nucleus, and arcuate nucleus, respectively. Pituitary stalk transection and hypophysectomy in Sprague-Dawley rats decreased insulin-like growth factor 2 and somatostatin mRNA and increased growth hormone-releasing factor mRNA. In each case, the effect of hypophysectomy exceeded that of pituitary stalk transection. Treatment with propylthiouracil for 10 days decreased somatostatin mRNA, markedly increased growth hormone-releasing factor mRNA but had no significant effect on insulin-like growth factor 2 mRNA. Treatment with triiodothyronine had no effect on the mRNAs measured. These findings corroborate the clinical observation of abnormal somatic growth in disturbances of thyroid and growth hormone status and provide further evidence of the effects of these metabolic disturbances and of pituitary disconnection and hypophysectomy on insulin-like growth factor 2 mRNA prevalence.


Dopamine-, NMDA- and sigma-receptor antagonists exert differential effects on basal and amphetamine-induced changes in neostriatal ascorbate and DOPAC in awake, behaving rats.

Amphetamine and other dopamine agonists elevate the extracellular level of neostriatal ascorbate, which has been shown to modulate neuronal function. To assess the receptor mechanisms underlying neostriatal ascorbate release, drug-induced changes in both basal and amphetamine-induced ascorbate release were monitored voltammetrically in the neostriatum of freely moving rats. A variety of dopamine receptor antagonists decreased basal ascorbate and reversed the increase induced by 2.5 mg/kg D-amphetamine. Thus, compared to vehicle treatment, administration of classical (haloperidol) and atypical (clozapine) neuroleptics or selective D1 (SCH-23390) and D2 (sulpiride) antagonists completely reversed the amphetamine-induced rise in ascorbate and also lowered basal levels by 20-40%. These same effects occurred following injection of dizocilpine (MK-801), a non-competitive NMDA antagonist, whereas BMY-14802, a sigma ligand, reversed the amphetamine-induced rise without altering basal levels. Simultaneous measurements of extracellular DOPAC, a major dopamine metabolite, revealed that haloperidol, clozapine, sulpiride and BMY-14802 elevated basal levels and reversed the amphetamine-induced decline. Dizocilpine also increased basal DOPAC but failed to alter the DOPAC response to amphetamine, whereas both basal and amphetamine-induced changes in DOPAC were unaffected by SCH-23390. A combination of subthreshold doses of SCH-23390 and sulpiride, however, reversed both the amphetamine-induced release of ascorbate and the corresponding decline in DOPAC. Collectively, these results suggest that whereas dopamine, sigma, and NMDA receptors modulate neostriatal ascorbate release, they exert an opposing influence on extracellular DOPAC. All drugs attenuated at least some components of the amphetamine behavioral response, suggesting a role for multiple mechanisms in the behavioral effects of this drug.


Changes in neurotransmitter parameters in the brain induced by L-cysteine injections in the young rat.

A single subcutaneous injection of L-cysteine (1.2 mg/g body wt.) to 4-day-old rats leads to atrophy of the brains examined 27-31 days later. The brains could be separated into two groups (type 1 and 2) on account of the degree of atrophy. Type-1 lesion, with a brain weight reduction of 20%, was dominated by a severe reduction in high-affinity uptake of L-glutamate in CNS regions receiving corticofugal fibers such as thalamus and striatum. Glutamate decarboxylase was only reduced in cortical structures. In type-2 lesion, with a severe brain atrophy of about 50%, high-affinity glutamate uptake was further reduced and there was a more pronounced reduction in glutamate decarboxylase activity in several brain regions. Cholinergic neurons were less affected by the lesion and the levels of choline acetyltransferase showed a relative increase in brain regions which partly compensated for their reduction in size.


Cell proliferative activity in adenomatous hyperplasia of the liver and small hepatocellular carcinoma. An immunohistochemical study demonstrating proliferating cell nuclear antigen.

Recently, adenomatous hyperplasia (AH) of the liver, a sizable parenchymal nodule in the cirrhotic liver, has been considered a preneoplastic lesion in human hepatocarcinogenesis. The authors evaluated cell proliferative activity by immunostaining for proliferating cell nuclear antigen (PCNA) in AH (n = 30), small hepatocellular carcinoma (HCC) (n = 14), and their surrounding regenerative nodules (SRN). AH was categorized histologically as ordinary or atypical. Ordinary AH (n = 8) had no hepatocellular atypia, whereas atypical AH (n = 22) was composed of atypical hepatocytes that were equivocal as to benignity and malignancy. Three atypical AH contained overt malignant foci. The PCNA labeling index of ordinary AH was lower than that of SRN and the index of atypical AH was higher than that of SRN except in two cases. The PCNA labeling index of malignant foci within atypical AH was higher than that of nonmalignant areas of atypical AH and was similar to that of small HCC. For small HCC, the PCNA labeling index was much higher than that of SRN and correlated with small HCC grades. These data suggest that ordinary AH is a largely developed regenerative nodule with little proliferative activity and that it is not a preneoplastic lesion; the data also indicate that atypical AH has much proliferative activity, from which malignant foci with greater proliferative activity emerge. Atypical AH with or without malignant foci may represent an early stage of multi-step hepatocarcinogenesis in humans.


Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB.

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to cAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.


The effect of anti-adhesion molecule antibody on the development of collagen-induced arthritis.

In order to study how inflammatory cells including autoimmune lymphocytes interact with each other to develop collagen-induced arthritis (CIA), we injected monoclonal antibodies against mouse LFA-1 and ICAM-1 into DBA/1 mice immunized with type II collagen (CII). Both antibodies suppressed the development of CIA. These antibodies showed no effect on anti-CII antibody response, although they both significantly suppressed DTH response. It was suggested that anti-adhesion molecule antibodies suppress CIA mainly through their effect on cell-mediated immunity, without affecting humoral immunity under the conditions used.


Regulation of human cytolytic lymphocyte responses by interleukin-12.

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.


IL-3 dependence of a Thy-1lo, B220+, IL-3 receptor-positive antigen-specific DTH-initiating clone.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different antigen-specific Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This clone WP-3.27 produces an antigen-specific factor, OX-F, that acts in an Ag-specific manner to initiate DTH. The clone was phenotyped as a Thy-1+, B220+, CD3-, CD4-, CD8- cell. In this report, we further detail the characteristics of this unusual Ag-specific DTH-initiating cell clone. By flow cytometry analysis, WP-3.27 is Thy-1lo, Lyt-1+ (CD5+), but CD3-, TCR-alpha beta-, and TCR-gamma delta-. Moreover, WP-3.27 does not express surface immunoglobulins but expresses B220 (CD45RA), and also some macrophage markers such as Mac-1, F4-80, and MHC class II after gamma-IFN treatment. Interestingly, this clone also expresses IL-3 receptors (IL-3R) and not IL-2R. In addition to the Ag-specific DTH-initiating factor, WP-3.27 constitutively produces IL-3. Inhibition of proliferation of WP-3.27 with an anti-mouse IL-3 monoclonal antibody suggests that the clone WP-3.27 is IL-3-dependent, at least partially. WP-3.27 also constitutively produces IL-1 and IL-6, but not TNF-alpha. LPS activation of the clone resulted in a net increase of IL-1, IL-6, and TNF-alpha production. Thus, this Ag-specific DTH-initiating cell clone makes a unique set of cytokines. Northern blot analysis demonstrated that clone WP-3.27 transcribes mRNA encoding IL-1, IL-3, IL-6, and TNF-alpha, but not for TNF-beta (lymphotoxin). The nature of this unusual cell, which displays characteristics of more than one cell lineage, is discussed.


Radioisotopic assay of aspartate and alanine aminotransferase.

The activities of aspartate and alanine aminotransferases in biological samples were assessed through a novel and sensitive procedure, based on the conversion of [U-14C]2-ketoglutarate to L-[U-14C]glutamate. In human plasma, the generation of L-[U-14C]glutamate was proportional to the volume of plasma (20-60 microL) and to the length of incubation (30-90 min). The reaction velocity was related to the temperature with a Q10 close to 1.7 for aspartate aminotransferase and 2.0 for alanine aminotransferase. At 37 degrees C, the 95% confidence interval in healthy subjects ranged from 5.1-18.8 U/mL (mean value 11.9 U/L) for aspartate aminotransferase and from zero to 20.1 U/L (mean value 9.9 U/L) for alanine aminotransferase. The intra-assay coefficient of variation did not exceed 2.5%. The present method was also applied to homogenates prepared from rat pancreatic islets, liver, heart, parotid glands, and erythrocytes, using no more than 40 micrograms wet weight of tissue per sample, and could thus be used in small biological samples, such as those obtained by needle biopsy.


Pharmacological evaluation of two novel analogues of mianserin: 2-N-carboxamidinonormianserin (FCC5) and 2-N-carboxamidonormianserin (FCC13).

1. The pharmacological properties have been examined of FCC5 (2-N-carboxamidinonormianserin) and FCC13 (2-N-carboxamidonormianserin), two novel analogues of mianserin. 2. FCC5 or FCC13 (100 micrograms/kg, i.v.) caused long-lasting (greater than 1 h) abolition of 5-hydroxytryptamine (5-HT) and histamine-induced bronchoconstriction in the anaesthetized guinea-pig. Both analogues had no effect (up to 1 mg/kg, i.v.) on bronchoconstriction caused by acetylcholine (25-50 micrograms/kg, i.v.). 3. The pressor effects of 5-HT in pithed rats were significantly attenuated by FCC5 (0.1 mg/kg, i.v.) or FCC13 (0.5 mg/kg, i.v.). 4. Oedema in the rat hind paw caused by intraplantar 5-HT was inhibited by FCC5 (ID50 0.76 mg/kg, i.p.; 2.7 mg/kg, p.o.) or FCC13 (ID50 0.65 mg/kg, i.p.; 5.8 mg/kg, p.o.). 5. In the central nervous system (CNS), FCC13 caused antagonism of 5-HT activity. It inhibited: (i) L-5-hydroxytryptophan (L-5-HTP)-induced head twitches in mice (ID50 1.85 mg/kg, i.p.), (ii) fenfluramine-induced facilitation of flexor reflex activity (FRA) in spinalized decerebrate rats (SDR) (IC50 0.57 mg/kg, i.p.). 6. FCC5 (less than or equal to 30 mg/kg, i.p. and less than or equal to 3 mg/kg, i.p., respectively) had no effect in either test. In contrast to mianserin, it also had no overt central actions as (less than or equal to 30 mg/kg, i.p.) had no effect on: (i) morphine-induced catalepsy (MIC) or (ii) clonidine-induced facilitation of FRA in SDR. However, high doses of FCC13 inhibited MIC (ID50 20 mg/kg, i.p.), but had no effect on (ii) (less than or equal to 10 mg/kg, i.p.). 7. Thus, FCC5 and FCC13 are potent, orally active H1 and 5-HT receptor antagonists. However, in contrast to FCC13 and mianserin, FCC5 did not cause CNS-mediated effects.


Autacoids affecting vascular tone in the human fetal extracorporeal circulation.

1. The human fetal extracorporeal circulation is normally a vasodilated, low pressure system. 2. As this vasculature lacks innervation, autacoids have been postulated as being of great importance in controlling its tone. 3. This has now been confirmed by pharmacological in vitro techniques, particularly utilizing perfusion of the isolated umbilical cord and placental lobule. 4. The fetal umbilical-placental vessels are sensitive to a wide range of vasoconstrictor autacoids, some of which can cause intense vasospasm. 5. Thromboxane A2 receptors have been identified in both umbilical vein and placental villous vessels. 6. Prostacyclin and endothelial cell-derived relaxing factor (or nitric oxide) may be largely responsible for the low vascular resistance normally found in the fetal extracorporeal circulation. 7. Immediately after birth it is likely that stimuli such as cooling, stretching and handling of the umbilical cord cause release of eicosanoids and other autacoids from the vessels, leading to their complete closure.


Evolution of phenotypic memory T cells in HIV-1 infected infants and children.

Infants are reported to be devoid of memory T cells at birth but acquired them with time. A cross-sectional study of peripheral blood mononuclear cells from HIV-infected and uninfected infants and children that bear the CD4R0 antigen was undertaken to describe the development of memory T cells. Linear regression lines derived from the data revealed increasing percentages of memory CD4 and CD8 cells in the uninfected children. Memory CD4 cells in the infected children were detected at a frequency equal to or greater than that seen in uninfected children until 6 months of age but subsequently declined with age. In contrast, memory CD8 cells were found to be significantly increased in HIV-infected children early in life with a rate of increase similar to that seen in the uninfected population. This increase in memory CD8 cells may facilitate the early diagnosis of HIV infection.


Lung mechanics and oxygen consumption during spontaneous ventilation and severe heart failure.

The effects of acute heart failure on lung mechanics and oxygen consumption (VO2) during normocarbic spontaneous ventilation were studied in 21 anesthetized pigs. Heart failure severe enough to double oxygen extraction (O2ex) was induced with intravenous esmolol boluses and infusion. Compared to normal, the inspiratory elastic work of breathing (Wel) increased from 335 +/- 371 (mean +/- SD) to 559 +/- 48 mm Hg.ml (p less than 0.003) during heart failure, lung compliance (CL) fell from 121 +/- 144 to 22 +/- 15 ml/mm Hg (p less than 0.05), and respiratory power climbed from 140 +/- 200 to 245 +/- 214 mm Hg.ml.min-1 (p less than 0.002). These mechanical changes were accompanied by a decrease in both VO2 (221 +/- 61 to 191 +/- 50 mlO2/min, p less than 0.05) and oxygen delivery (DO2) (680 +/- 240 to 260 +/- 90 mlO2/min, p less than 0.004). The VO2/DO2 ratio doubled (p less than 0.0002), confirming increased O2ex. In conclusion, severe acute heart failure decreased CL, and increased Wel and respiratory power significantly. The depressed cardiac output limits both DO2, and to some extent, VO2. However, a greater proportion of the delivered O2 is consumed, supplying indirect evidence which suggests that the respiratory muscles' VO2 increases as a consequence of increased power expenditure.


Bronchiolitis obliterans associated with polyarteritis nodosa.

Bronchiolitis obliterans with organizing pneumonia (BOOP) has not previously been described in association with polyarteritis nodosa (PAN). This report describes a patient in whom fulminant systemic PAN followed subacute onset of BOOP, with associated pulmonary arteritis.


Comparison of casual, ambulatory and self-measured blood pressure in a study of nitrendipine vs bisoprolol.

In a double-blind, placebo-controlled study the antihypertensive efficacy and tolerability of a single morning dose of either 10 mg bisoprolol (n = 26) or 20 mg nitrendipine (n = 27) were investigated. Blood pressure was measured by three techniques: (1) Casual blood pressure 24 h after the dose; (2) ambulatory 24-h whole-day monitoring; and (3) self-recorded blood pressure in the morning 24 h after the dose (6-8 a.m.) and in the evening (6-8 p.m.). After 4 weeks of therapy bisoprolol had produced a highly significant reduction in blood pressure as assessed by causal, ambulatory day- and night-time monitoring, and self-measured morning and evening readings. Bisoprolol was significantly more effective than nitrendipine, which did not induce a significant reduction in the ambulatory night-time recordings. Whole-day ambulatory blood pressure profiles showed an antihypertensive effect of bisoprolol throughout the entire 24-h period. 24-h blood pressure curves after nitrendipine demonstrated a markedly shorter duration of action, with no reduction in early morning blood pressure. Adverse effects and tolerability of the two drugs were comparable. The average changes in systolic and diastolic blood pressure after bisoprolol and nitrendipine in 2-h periods of ambulatory monitoring (6-8 a.m. and 6-8 p.m.) and self-measured blood pressure (6-8 a.m. and 6-8 p.m.) showed a good agreement between ambulatory and self-measured blood pressure determinations with no significant difference between the methods. The results show that 24 h antihypertensive efficacy was more pronounced for bisoprolol than for nitrendipine at the doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)


Does treatment with beta-adrenergic blocking agents cause a decrease in beta 2-adrenoceptor affinity?

The effect of beta-adrenoceptor antagonists (BAAs) differing in lipophilicity and partial agonist activity (PAA), and a full agonist, on the dissociation constant for [125I]-(-)- iodocyanopindolol binding to beta 2-adrenoceptors (KD) has been investigated. Twelve healthy, normotensive male volunteers (mean age 22.3 y) were treated with different BAAs according to a cross-over design. The drugs used were propranolol (highly lipophilic BAA, no PAA), pindolol (moderately lipophilic BAA, strong PAA), dilevalol (highly lipophilic BAA, weak PAA) and salbutamol (full agonist). Before and after a single dose and an 8 day course of one of the drugs, blood pressure and the beta 2-adrenoceptor characteristics of mononuclear leukocytes (MNL) were determined. Between the treatment periods, there was a washout interval of 14 days. All BAAs decreased the blood pressure, but only propranolol lowered heart rate. Treatment with salbutamol decreased the diastolic and increased the systolic blood pressure and heart rate. Three hours after the single dose of any of the BAAs, a more than 2-fold increase in KD was observed, and the increase became larger after 8 days of administration (up to 3.7-fold increase). In contrast, no effect on KD was observed after treatment with salbutamol. BAAs with PAA and salbutamol induced a 30% decrease in beta 2-adrenoceptor density. It is concluded that treatment with BAAs, irrespective their lipophilicity or PAA, induces a decrease in the affinity of MNL beta 2-adrenoceptors for antagonists. This phenomenon may help to explain the contradictory relationship between the kinetics and dynamics of BAAs.


Minimal growth requirements of mature T lymphocytes: interleukin (IL)-1 and IL-6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti-CD3 and IL-2.

We show that interleukin (IL)-2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic-bound anti-CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL-1 or IL-6, alone or in combination. These cytokines were unable to induce responsiveness to IL-2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL-1 and IL-6 increase the growth rate of CD4 cells. The addition of IL-6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL-2 alone, p less than 0.01), while the addition of IL-1, ineffective by itself, combined with IL-6 further increased the growth rate of CD4 cells (dt: 23 h, p less than 0.001). The growth rate of CD8 cells stimulated with anti-CD3 and IL-2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p less than 0.001) and was not significantly influenced by addition of IL-1 and/or IL-6.


Induction of homotypic T cell adhesion by triggering of leukocyte function-associated antigen-1 alpha (CD11a): differential effects on resting and activated T cells.

The leukocyte integrin LFA-1 (CD11a/CD18) plays a key role in many adhesive interactions involving cells of the immune system. Recently, it has been shown that LFA-1 is not only involved in cell adhesion, but that stimulation of LFA-1 can also contribute to cell activation. We now demonstrate that triggering of LFA-1 on T lymphocytes by monoclonal antibodies (mAb) against the LFA-1 alpha chain, but not against the LFA-1 beta chain, promotes cell adhesion. Induction of homotypic adhesion was only observed in T cells that had been pre-activated with anti-CD3 and not in resting peripheral blood T lymphocytes. The induced homotypic adhesion is mediated by LFA-itself, because it was inhibited by anti-LFA-1 beta mAb. This notion is supported by the temperature and divalent cation dependence which is characteristic of LFA-1-mediated adhesion. mAb against ICAM-1 (CD54) did not block LFA-1 alpha-induced adhesion. The sensitivity of LFA-1 alpha-induced adhesion to H7, which prevents the activation of protein kinase C and protein kinase A, and to cytochalasin B, which inhibits microfilament formation, suggests that the activation of the LFA-1 pathway through the LFA-1 alpha chain involves cell activation and requires an intact cytoskeleton.


Four CD45/P56lck-associated phosphorproteins (pp29-pp32) undergo alterations in human T cell activation.

In human T lymphocytes a functional complex is formed between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and a phosphoprotein, pp32, a possible common substrate. Here we demonstrate that the previously described pp32 protein is composed of two distinct molecules (pp29 and pp32) in both resting human T lymphocytes and continuously proliferating T lymphoma lines. Importantly, T lymphocyte activation employing CD2 monoclonal antibodies (mAb), CD3 mAb or phorbol 12, 13 dibutyrate results in loss of pp29 and pp32 from the CD45/p56lck molecular complex and concomitant association of two distinct phosphoproteins with different molecular weights (pp30 and pp31). These events appear to be unrelated to clonal T cell growth but rather depend on receptor-mediated differentiation signals. Reprecipitation experiments employing an antiserum directed at a consensus sequence of GTP-binding proteins suggest that all four pp29-pp32 molecules might represent proteins with GTP-binding properties. Biochemical analysis of pp29-pp32 employing V8-protease digestion indicates that they differ in low-molecular weight fragments of 8, 5, 4.5, 4 and 3 kDa, respectively.


Molecular basis for a severe case of leukocyte adhesion deficiency.

The leukocyte integrins LFA-1, Mac-1 and p150,95 (CD11a/CD18, CD11b/CD18, CD11c/CD18) mediate crucial leukocyte adhesive functions in immune and inflammatory reactions. Leukocyte adhesion deficiency (LAD) disease is caused by the defective expression of these adhesion molecules on leukocytes, and is characterized by recurrent infections and impaired pus formation due to the blockade of leukocyte migration into inflamed tissues. LAD is originated by heterogeneous mutations affecting the CD18 gene and, based on the severity of the deficiency, two phenotypes (severe and moderate) have been defined. Biochemical and genetic studies have allowed the classification of five different types of LAD. We have identified a type V LAD patient (severe phenotype, and normal size and levels of both CD18 precursor and CD18 mRNA), and determined its molecular basis. Reverse transcription-polymerase chain reaction and cloning and sequencing of CD18 cDNA derived from this patient revealed three silent mutations and a missense mutation that leads to the substitution of glycine at position 169 for an arginine. Analysis of patient-derived cDNA clones revealed the concomitant presence of aberrant splicing within the 5' region of the CD18 gene. The description of an identical mutation at residue 169 in an unrelated severe LAD patient raises the possibility that severe LAD type V is caused by a unique genetic defect.


Genetic control of nonresponsiveness to hepatitis B virus vaccine by an extended HLA haplotype.

We previously reported evidence for a statistical association between the serologically determined HLA-Bw54, DR4 and DRw53 alleles and the non-immune responsiveness to hepatitis B virus surface antigen (HBsAg) in the Japanese population. To identify the locus and allele within the HLA region associated with the nonresponsiveness to HBsAg, serological HLA typing, DNA typing of HLA-DQ and DP alleles using amplified HLA genes and sequence-specific oligonucleotide probes, and restriction fragment length polymorphism (RFLP) analysis of the fourth component of complement (C4) genes were performed in healthy unrelated Japanese vaccinees who were immunized subcutaneously three times with plasma-derived HBsAg vaccine. In nonresponders to HBsAg, the frequencies of HLA-Bw54 cross-reactive epitope group (CREG); (Bw54, Bw55, Bw56 and other Bw22), C4 RFLP (6.5 kb + 12.0 kb), DR4, DRw53 and DQw4 (DQA1*0301-DQB1*0401) were increased and the frequencies of HLA-DR1, DRw6 and DQw1 were decreased as compared with those in healthy unrelated controls. Further analysis revealed that the coexistence of HLA-Bw54CREG and DR4-DRw53-DQw4 (DQA1*0301-DQB1*0401) was associated with the nonresponder group, whereas, donors positive for exclusively either Bw54 CREG or DR4-DRw53-DQw4 (DQA1*0301-DQB1*0401) were not associated with the nonresponder group. Because there is a strong linkage disequilibrium between HLA-Bw54CREG, C4 RFLP (6.5 kb + 12.0 kb) and HLA-DR4-DRw53-DQw4 (DQA1*0301-DQB1*0401) in the Japanese population, the extended HLA-Bw54CREG-C4 RFLP (6.5 kb + 12.0 kb)-DR4-DR-w53-DQw4 (DQA1*0301-DQB1*0401) haplotype may well control nonimmune responsiveness to HBsAg. This extended HLA haplotype controls nonresponsiveness as a dominant genetic trait because all ten heterozygotes and two of three probable homozygotes of this extended HLA haplotype were nonresponders.


Effect of apraclonidine on blood-aqueous barrier permeability to plasma protein in man.

Effect of a single instillation of 30 microliters 0.5% apraclonidine on the coefficient of protein entry (k(in)), a quantitative index of blood-aqueous barrier permeability, was determined in ten normal volunteers. Before and after administering the drug to one eye and the vehicle to the other eye of a subject, protein concentrations in the anterior chamber were measured using a laser flare-cell meter. One week later, aqueous flow rate was determined by fluorophotometry under the same experimental conditions. The k(in) was calculated from protein concentrations in the anterior chamber and the plasma and from aqueous flow rate based on a transfer equation formulating the kinetics of protein molecules in the anterior chamber. The k(in) showed a significant reduction at 3 and 4 hr after drug administration with a maximum decrease of 21 +/- 6% (mean +/- S.E., n = 10) at 3 hr, while the aqueous flow rate showed a significant reduction from 2 to 6 hr with a maximum decrease of 45 +/- 5% at 3 hr. The results indicated that topical apraclonidine reduced both blood-aqueous barrier permeability as well as aqueous flow rate in humans. This dual reduction may relate to the efficacy of this drug in suppressing the post-laser intraocular pressure rises in patients.


Quinpirole alters quadruped activity in rats from the second postnatal week.

The present study shows that the effects of quinpirole (1 mg/kg) during the second postnatal week but not before resemble the effect of the drug in adult rats in increasing quadruped activity, eliminating grooming, reducing lateral bending, and stimulating verticalized turning. Quinpirole also modifies the morphology of turning behavior, the primary form of coordinated quadruped locomotion in neonate laboratory rat pups. Under saline, turning involves lateral bending and straightening of the trunk. Under quinpirole, turning includes a vertical component of movement ("verticalized turning") instead of the normal lateral bending of the trunk. A similar trend of change in turning is induced by quinpirole in adult rats: An acute injection reduces lateral bending, and a chronic treatment increases verticalization. The induction of vertical turning in the second but not the first postnatal week may stem from the normal course of development since typical vertical movements of intact rats (supported rearing and wall climbing) develop only by the age of 11-14 postnatal days. Verticalized turning may be thus a drug-induced expression of an age-related tendency to perform vertical movements.


An experimental study on the gastric acid and gut hormone secretion after pylorus preserving duodenectomy in dogs.

To clarify changes in gastric acid and gut hormone secretion after pylorus-preserving pancreaticoduodenectomy (PPPD), an experimental study was performed using a model of pylorus-preserving duodenectomy in dogs previously provided with Heidenhain pouch (HP). The duodenectomy involves resection of the duodenum and 10 cm of the proximal jejunum preserving 2 cm of juxtapyloric duodenum and round-shaped duodenal wall around pancreatic papilla. Reconstruction was done by anastomosing the rho-shaped jejunal loop to gallbladder, juxtapyloric duodenum and peripapillar round-shaped duodenal wall with ligation of the common bile duct. For these dogs, intravenous glucose tolerance test (IVGTT), oral glucose tolerance test (OGTT), meal ingestion test (TM) and histological studies of pancreatic specimen obtained at autopsy were performed investigating pancreatic, gastric acid and gut hormone secretion. Preservation of endocrine and exocrine pancreatic secretion after operation demonstrated our experimental model to be adequate for evaluation of the factor of duodenectomy in PPPD on gastric acid and gut hormone secretion avoiding the influences of changes in pancreatic secretion. Postprandial gastric acid secretion from HP did not change significantly after operation. Postprandial secretion of gastrin, glucagon, GIP and enteroglucagon did not alter significantly after operation. These results indicated that in the clinical PPPD procedure, preservation of more than 2 cm of duodenum from the pylorus produced neither postprandial gastric acid hypersecretion, which might be cause of postoperative stomal ulcer, nor any change of related gut hormone secretion.


Effects of converting enzyme inhibition and alpha receptor blockade on the angiotensin pressor response in the American alligator.

This study examines the effects of two converting enzyme inhibitors (captopril and enalaprilat) and two alpha-adrenergic receptor antagonists (phentolamine and phenoxybenzamine) on the pressor response produced by exogenous angiotensin I ([Asp1, Val5, Ser9] ANG I, fowl) and [Val5] angiotensin II (ANG II) in the American alligator (Alligator mississippiensis). Bolus administration of ANG I at 0.1, 0.5, and 1.0 micrograms/kg; ANG II at 0.05, 0.1, and 0.5 micrograms/kg; or norepinephrine (NE) at 2 micrograms/kg elicited dose-dependent increases in arterial blood pressure. Captopril (0.5 mg/kg/hr) and enalaprilat (300 micrograms/kg/hr) significantly reduced the response to ANG I, but not ANG II or NE. Both phenoxybenzamine (0.25 mg/kg/min) and phentolamine (1 mg/kg/hr) effectively blocked the NE pressor response (84 and 88%, respectively) and attenuated (42-80%) the pressor effects of ANG I and ANG II. These results support previous work suggesting the alligator may possess a renin-angiotensin system with characteristics similar to those found in mammals and other vertebrates. In addition, the pressor response to exogenously administered ANG I and ANG II was attenuated by alpha adrenergic receptor blockade and thus may be due, in part, to secondary catecholamine release.


Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin.

The mature forms of the extracellular muramidase-2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active-site-containing N-terminal domain fused to a multiple-repeat C-terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.


Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting.

DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata. Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species. The strains used in this study differ only in terms of their geographic origin or genetic background. One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13. Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction. Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic. Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30%. The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.


Vitamin A deficiency and colonic electrogenic absorption and secretion in the rat.

The effects of vitamin A deficiency on electrogenic transport in the colon were examined in rats made vitamin A deficient at weaning by feeding a vitamin A deficient diet for 40 days. A pair fed control group was given the same diet but supplemented with soluble vitamin A in their drinking water. The basal and stimulated electrogenic secretory and absorptive functions of the muscle stripped proximal, mid, and distal colon were examined in vitro using the short circuit current (Isc) as the index of net ion transport. A significant increase in the basal and secretory Isc (mainly Cl-ions) induced by the cholinergic agonist bethanechol was observed in the mid-colon of the vitamin A deficient rats. In the distal colon, however, vitamin A deficiency caused a significant reduction in both the basal and secretory Isc response to bethanechol compared with the vitamin A supplemented pair fed control. Secretory Isc induced by dibutyryl cyclic adenosine monophosphate was not significantly altered by vitamin A deficiency. The condition abolished the response of the distal colon to luminal amiloride (0.1 mmol/l). Thyroid hormone induced reduction in the distal colonic response to aldosterone is implicated in this lack of response. This is the first experimental linkage between vitamin A action, the thyroid hormone and aldosterone on colonic function. The colonic changes induced by vitamin A deficiency, namely hypersecretion and a reduced electrogenic distal absorptive function, together with the previously described small intestine hypersecretion may be the underlying basis for the diarrhoea observed in human and animal vitamin A deficiency.


Mechanism of action of an antifungal antibiotic, RI-331, (S) 2-amino-4-oxo-5-hydroxypentanoic acid; kinetics of inactivation of homoserine dehydrogenase from Saccharomyces cerevisiae.

An antifungal antibiotic (S) 2-amino-4-oxo-5-hydroxypentanoic acid, inhibited the biosynthesis of the aspartate family of amino acids (methionine, isoleucine and threonine) followed by the inhibition of protein biosynthesis in Saccharomyces cerevisiae. This inhibition was effected by impeding the biosynthesis of their common intermediate precursor, homoserine. The inhibition of biosynthesis of homoserine by the antibiotic was attributable to inactivation of homoserine dehydrogenase [EC 1.1.1.3], which is involved in the conversion of aspartate semialdehyde to homoserine in the metabolic pathway leading to threonine, methionine and isoleucine. Since such enzymic activity is not present in animal cells, the selective antifungal activity of the antibiotic is thus explained.


Characterization of Escherichia coli glnL mutations affecting nitrogen regulation.

Nitrogen regulator II (NRII), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI. Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NRI-phosphate phosphatase activity that is observed when NRII and another signal transduction protein known as PII (the glnB product) interact. The availability of PII for this interaction is controlled by a uridylytransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies PII in response to intracellular signals of nitrogen availability. Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation. The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains. Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NRII autophosphorylation at His-139. Complementation experiments with multicopy plasmids encoding NRII or PII showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NRII but was not affected by introduction of the plasmid encoding PII. Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NRII or that encoding PII. We hypothesize that mutants of the latter type result in a subtle perturbation of the NRII-PII interaction and suggest two possible mechanisms for their effects.


DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology.

Mycobacterium tuberculosis isolates were studied by comparing large restriction fragment (LRF) patterns produced by digestion of chromosomal DNA with infrequent-cutting endonucleases and pulsed-field gel electrophoresis. Four cultures of H37Rv and 36 clinical isolates of M. tuberculosis were compared by using DraI, AsnI, XbaI, and SpeI. DraI and AsnI allowed easy visual separation of 18 of 21 epidemiologically unrelated strains. XbaI and SpeI allowed discrimination of all 21 unrelated strains, including the 3 strains inseparable with DraI and AsnI, but comparison of LRF patterns was more tedious because of overlapping fragments. A total of 26 isolates belonging to 10 clusters of related isolates were compared by pulsed-field gel electrophoresis, with all related isolates giving identical LRF patterns. These included multiple isolates from the same patient or the same family. The same grouping of clustered isolates was obtained when BamHI DNA digests were hybridized with two probes from the insertion sequence IS6110. Long-term laboratory passage of H37Rv produced minimal detectable changes in LRF patterns. LRF patterns are useful tools for epidemiologic studies of tuberculosis without the need for radioactive or specific DNA probes.


Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii.

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.


Cerebrospinal fluid neuropeptides in mood disorder and dementia.

Cerebrospinal fluid (CSF) concentrations of immunoreactive corticotropin-releasing hormone (CRH) and somatostatin (SRIF) were measured in female psychiatric inpatients with DSM-III-R diagnoses of major depression, mania, generalized anxiety and somatization disorder. In addition, elderly patients with dementia disorders, with or without concomitant major depression, were also investigated. CSF SRIF was not significantly different among these groups; on the other hand, mean CSF CRH concentrations were significantly higher in major depression and in dementia with depression as compared with neurological controls with no psychiatric disorders. CSF CRH levels in mania, simple dementia, or anxiety or somatization disorder were not significantly different from the controls. Background physical or clinical variables did not account for the differences in CRH concentrations. It is concluded that CSF CRH elevation may be present in some patients with major depression independent of age and an underlying dementia disorder.


Zotepine in the treatment of schizophrenic patients with prevailingly negative symptoms. A double-blind trial vs. haloperidol.

Zotepine, a neuroleptic agent with additional 5-HT2 blocking properties, was compared with haloperidol in the treatment of schizophrenic patients with predominantly negative symptoms using a double-blind design. During the investigation period zotepine treated patients showed significant improvements in all rating instruments whereas haloperidol treated patients did not. Patients in the zotepine group developed fewer clinical side effects. The results of the presented study confirm the positive impressions gained in earlier open trials with zotepine.


Thymocyte costimulating antigen is CD26 (dipeptidyl-peptidase IV). Costimulation of granulocyte, macrophage, and T lineage cell proliferation via CD26.

The rat thymocyte costimulating protein appears to be involved in the regulation of CD4-/CD8- thymocyte proliferation in vitro. We show that thymocyte costimulating protein is dipeptidyl peptidase IV (E.C.N.3.4.14.5) also known as CD26. Some bone marrow cells as well as CD4-/CD8- and, and to a lesser extent, more differentiated T cells respond by proliferating to a CD26 specific mAb-mediated costimulus that does not influence dipeptidyl dipeptidase IV enzyme activity. This suggests the existence of a CD26-linked regulatory mechanism of proliferation that is operational on granulocyte- and macrophage-lineage cells and throughout T cell development.


Contributions of the CD2 and CD28 T lymphocyte activation pathways to the regulation of the expression of the colony-stimulating factor (CSF-1) gene.

The T cell adhesion molecule CD28 provides a costimulatory signal, in combination with either CD2 or CD3 mAb. CD28 appears to regulate the expression, by T cells, of cytokines normally produced by accessory cells, namely IL-1 alpha, TNF-alpha, and CSF-1. The CSF-1 gene is expressed as a 4.0-kb transcript by human T cells activated with mAb directed against CD2 and CD28, alone or in combination. A kinetic analysis of its expression showed low steady-state levels of the transcript after CD2 stimulation, and a transient rise after CD28 stimulation. In contrast, a mean fivefold increase in the levels of the transcript was detected in CD2 plus CD28-stimulated cells. The potential mechanisms regulating this increase were investigated. The half-life of the CSF-1 transcript was identical in cells activated with either CD28 or CD2 plus CD28. Transcription of the CSF-1 gene appeared to undergo attenuation in resting cells as well as in cells activated via a single pathway; this attenuation was relieved in part by the combined CD2 plus CD28 stimulation. Thus in vitro costimulation via the CD2 and CD28 molecules regulates the expression of the CSF-1 gene mainly at the transcriptional level.


The mtDNA genealogy of closely related Drosophila silvestris.

Genetic, morphological, and behavioral analyses have been used to examine the evolutionary dynamics and phylogeny of the rare Hawaiian Drosophila species, D. silvestris. Critical to understanding the evolution of this species is the examination of the distribution of populations of D. silvestris on the Big Island of Hawaii. Behavioral analysis using mating asymmetries and the Kaneshiro hypothesis as an indicator of ancestral behavioral state has suggested that flies from the northern part of the island are ancestral to those on the southern part of the island. Consequently, a sequential pattern of colonization going from north to south is predicted for these flies on the east side of the Island of Hawaii. We have examined this prediction using mitochondrial DNA (mtDNA) restriction site analysis with four-base cutters and DNA sequencing. The resulting mtDNA phylogeny based on 23 phylogenetically informative restriction sites and two phylogenetically informative DNA sequence characters agrees in part with the phylogeny predicted from the behavioral data.


Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. I. Long term augmentation by transient increases in intracellular cAMP.

Lymphocyte adhesion to target cells is mediated, in part, by the interaction of lymphocyte function-associated Ag-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1). Cells of the B cell line, JY, express both coreceptors and have been used as a model for intercellular adhesion mediated by these molecules. Elevation of the intracellular cAMP concentration ([cAMP]i), by any of several reagents, for periods as brief as 30 min, led to enhanced intercellular adhesion in a concentration dependent manner 5 to 8 h later. Two protein kinase A inhibitors, KT5720 and H-89, but not the protein kinase C inhibitor calphostin C, blocked the effects of elevated [cAMP]i. These data suggest a role for protein kinase A in this response. The adhesion augmented by increased [cAMP]i was due to LFA-1/ICAM-1 interactions between cells because it was blocked by either anti-LFA-1 or anti-ICAM-1 mAb. Elevated [cAMP]i induced cell surface patching of LFA-1, but not ICAM-1, and this redistribution preceded intercellular adhesion. Finally, redistribution of LFA-1 was not mediated by the cytoskeleton. These results suggest a model in which transient activation of protein kinase A results in increased local concentration of LFA-1 at the cell surface and in augmented long term adhesion mediated by this integrin.


Lymphocyte adhesion mediated by lymphocyte function-associated antigen-1. II. Interaction between phorbol ester- and cAMP-sensitive pathways.

Ag independent adhesion between lymphocytes and target cells is mediated in part by the interaction between lymphocyte function associated Ag-1 (LFA-1) and its coreceptor intercellular adhesion molecule-1 (ICAM-1). Within minutes, PMA treatment of JY cells, which express both LFA-1 and ICAM-1, induced capping of LFA-1 and augmentation of intercellular adhesion lasting for several hours. However, over the course of 15 to 30 min, both of these events were blocked by elevation of intracellular cAMP concentration ([cAMP]i) presumably via activation of protein kinase A. This short term inhibition of protein kinase C-induced adhesion was in contrast to the long term augmentation of adhesion caused by increased [cAMP]i as demonstrated in the companion article. Intercellular adhesion, due to LFA-1/ICAM-1 interactions, could also be induced by LPS treatment of JY cells. At submaximal concentrations, the extent of aggregation induced by LPS had two maxima, one at 30 to 60 min and the other with a plateau at 5 to 8 h. LPS is known to activate protein kinase C and we show that LPS treatment induced increased [cAMP]i. Using inhibitors of protein kinases C and A, possible mediators of the two components of adhesion induced by LPS could be identified. The early component was abrogated by inhibition of protein kinase C although the later component was unaffected. In contrast, an inhibitor of protein kinase A had no affect on the early component and attenuated, but did not entirely eliminate, the late component. These results suggest a model of sequential induction, inhibition, and reinduction of LFA-1/ICAM-1-mediated lymphocyte adhesion that is regulated by temporally ordered actions and interactions of protein kinases C and A.


Instability of the minisatellite sequence in the first intron of the rat renin gene and localization of the gene to chromosome 13q13 between FH and PEPC loci.

A minisatellite sequence in the first intron of the rat renin gene showed five-allelic polymorphism in 11 inbred rat strains. A new allelic variant, which was thought to be generated in the germ line, was observed in 136 animals of two sets of backcross progenies originating from parental strains with different alleles. These facts suggested that the minisatellite is genetically unstable. A linkage analysis using the backcross progenies confirmed the assignment of renin locus (REN) on linkage group (LG) X at a site between FH (fumarate hydratase) and PEPC (peptidase) loci. Fluorescence in situ hybridization allowed mapping of the renin gene on rat chromosome 13q13.


Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity.

The existence of CD3/TCR-bearing lymphocytes in athymic and thymectomized chimeric mice implies that T cell maturation can occur in the absence of a thymus. Considering the possibility that the epidermis may be one of the organs providing T cell educating stimuli, we attempted to characterize the Thy-1+ epidermal lymphocyte population of athymic mice. Immunohistologic studies of epidermal sheets revealed (1) that Thy-1+ epidermal cells of C57BL/6 nu/nu mice are CD5-, CD4-, and predominantly CD8-, and (2) that a minor subset of these cells displays anti-CD3 epsilon reactivity. Although these CD3+ epidermal cells could hardly be detected at 6 wk of age, they comprised approximately 2% of all Thy-1+ epidermal cells in 12-mo-old athymic mice. Most of these CD3+ cells expressed TCR-gamma/delta, but TCR-alpha/beta+ cells were also present. TCR-gamma/delta+ epidermal T cells of athymic mice preferentially expressed TCR V gamma 2, V gamma 4, and V gamma 5 specificities rather than TCR V gamma 3 as found on DETC of euthymic mice. Using mitogenic stimuli, we have succeeded in establishing cell lines and clones from BALB/c nu/nu and C57BL/6 nu/nu epidermis. Their marker profile corresponds to that seen on resident CD3+ epidermal cells, as well as on a very small subset of CD3+ splenic and lymph node lymphocytes of athymic mice. The ontogenetic relationship, if any, between the epidermal and lymphoid CD3+, CD5-, CD4-, CD8- cells, has yet to be clarified. Cell lines/clones representative of resident CD3+ epidermal cells of nu/nu mice should provide a useful tool in the elucidation of homing patterns and functional properties of extrathymically matured T cells.


Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.


Sequential analysis of T cells in the liver during murine listerial infection.

Phenotypes and functions of T cells in the liver were studied after an i.p. inoculation with viable Listeria monocytogenes in mice. T cells in the liver of untreated C3H/HeN mice (C3H; H-2k, Mls-2a) contain Thy-1.2+TCR-alpha beta + cells as a majority and Thy-1.2+TCR-gamma delta + cells and Thy-1.2-TCR-gamma delta + cells as minorities. The liver of untreated C3H mice did not contain T cells expressing V beta 3 and V beta 11, which are potentially autoreactive against self-superantigens of Mls-2a and Dvbl, respectively. On days 3 to 6 after infection, Thy-1.2-CD4lowTCR-alpha beta + T cells or Thy-1.2-TCR-gamma delta + T cells increased significantly in number and proportion in the liver whereas T cells with these phenotypes were hardly detected in the spleen, lymph nodes, peripheral blood, and peritoneal cavity during the course of the infection. The Thy-1.2-CD4lowTCR-alpha beta T cells contained V beta 3 or V beta 11-bearing cells in high frequencies. The potentially autoreactive V beta 3- or V beta 11-bearing T cells disappeared from the liver on day 7 after infection. Furthermore, the V beta 3+ and V beta 11+ cells but not V beta 8+ cells disappeared after culture for 24 h at 37 degrees C. In vitro stimulation of liver T cells using anti-V beta 11 mAb showed no proliferative response. These results suggest that the potentially autoreactive clones with Thy-1.2-CD4low phenotypes, which increased in number after listerial infection, may be anergized after interaction with self-Ag and may be programmed to die. These potentially autoreactive clones induced in the liver of Listeria-infected mice may not be functionally relevant to the host defense against Listeria.


Human myoblasts as antigen-presenting cells.

Human myoblasts, cultured from muscle and purified to greater than 95%, were investigated for their capacity to act as facultative APC. The myoblasts reacted with antidesmin mAb and had the capacity to fuse into multinucleated myotubes in appropriate medium. The expression of HLA class I, HLA-DR, HLA-DP, HLA-DQ, intercellular adhesion molecule-1 (ICAM-1/CD54), lymphocyte function-associated (LFA) molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) was investigated by FACS analysis before and after induction for various times with human rIFN-gamma, TNF-alpha, or both. Without cytokine induction, myoblasts expressed only HLA-class I and LFA-3. IFN-gamma alone or in combination with TNF-alpha induced the expression of HLA-DR and ICAM-1 reaching a plateau after 48 h, followed by HLA-DP and even later HLA-DQ. TNF-alpha alone induced only ICAM-1. The functional capacity of myoblasts to present Ag to CD4+ T cells was investigated using autologous T cell lines specific for tuberculin, tetanus toxoid, and human myelin basic protein. Noninduced myoblasts or myoblasts treated with TNF-alpha alone could not present any of these Ag to the T cells. However, myoblasts treated with IFN-gamma induced Ag-specific proliferation. In the presence of relevant Ag, myoblasts were killed by the T cells as observed by microscopy and measured by 51Cr release. Ag-specific T cell proliferation and myoblast killing was inhibited in the presence of anti-DR mAb. These results suggest that human myoblasts may act as facultative APC during local immune reactions in muscle.


Sufentanil: clinical use as postoperative analgesic--epidural/intrathecal route.

Although morphine and fentanyl remain the predominant epidural opioids, sufentanil offers some unique advantages. Because of its greater lipophilicity and mu-receptor binding capacity, sufentanil has a faster onset of action and longer duration than epidural fentanyl. Compared with morphine, sufentanil has been associated with a lower incidence of side effects, particularly delayed respiratory depression. The effective doses and adverse effects profile of epidural sufentanil are relatively well understood. Ventilatory depression is minimal with both bolus and continuous administration. Rapid vascular uptake after large epidural bolus, however, has been associated with acute-onset respiratory depression and even respiratory arrest. Sufentanil is more ideally suited than morphine to continuous epidural administration. The faster onset in comparison with fentanyl may make sufentanil the ideal agent for patient-controlled epidural analgesia. The synergistic effect of combined sufentanil and low-concentration bupivacaine offers advantages over sufentanil alone. High doses of epidural sufentanil have been uniquely associated with cessation of shivering and hypothermia. As with fentanyl, the intrathecal administration of sufentanil for postoperative analgesia is limited by its short duration of action.


Molecular genetics of Leber's hereditary optic neuropathy: study of a six-generation family from Western Australia.

Molecular genetic studies were carried out on a 6-generation family from Western Australia with Leber's hereditary optic neuropathy. Pedigree analysis confirms the maternal inheritance of the genetic lesion underlying the disorder in this family. The presence of a recently reported disease-associated mutation at nucleotide 11778 of the mtDNA was established in one clinically affected family member by the sequencing of an appropriate 1.6 kb PCR-amplified fragment of the mtDNA; this mutation leads to an Arg340----His amino acid replacement in the ND4 subunit of respiratory complex I. The 11778 G to A base substitution is associated with the loss of an SfaNI restriction site. Examination of the representative members for this site revealed that while only mtDNA carrying this substitution could be detected in the leukocytes of 4 family members of the sixth generation, the mutated mtDNA was found to co-exist with the normal mtDNA population (heteroplasmy) in a clinically unaffected member from the fifth generation. This observation suggests that the nt 11778 mutation observed in this LHON family is relatively new; the observation of both heteroplasmy and apparent homoplasmy of the mtDNA in different family members might reflect the normal progression in the establishment of a mitochondrially inherited mutation.


T-cell phenotypic profiles in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients.

Thirty-nine patients with clinically definite multiple sclerosis (MS) entered the study. Of 28 subjects with a relapsing-remitting course, 19 were classified in acute relapse, 9 in remission; 11 patients had a progressive course without remissions. Furthermore, 6 subjects with inflammatory neurological disease (IND), and 10 with non-inflammatory and non-neoplastic neurological disease (NIND) were investigated. We simultaneously studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-, B- and NK-cell subsets, as defined by following monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD19, -CD16, -HLA-DR and -IL-2-R. We found a significant increase of CD4+ T-cells compared with controls in CSF, with respect to PB, of MS patients, particularly in acute relapse. An increase of HLA-DR+ cell percentages in the CSF than in the PB in all MS groups, especially in attacks of MS but also in remission, was also observed, with a positive correlation between CD4+ T-cell and DR+ cell percentages both in the CSF as well as in the PB of relapsing MS patients. These findings, together with the increase of IL-2-R+ cells in the PB, particularly in relapsing MS, give further support for the presence of a systemic T-cell activation in MS.


The probable conformation of substrates recognized by dipeptidyl-peptidase IV and some aspects of the catalytic mechanism derived from theoretical investigations.

By theoretical conformational investigations of substrates and nonsubstrates of the enzyme dipeptidyl-peptidase IV (DP IV) as well as dipeptide-esters using the ECEPP83 method we determined the structure of peptides recognized and cleaved by the enzyme. From a comparison of all possible structures for the substrates with conformations not possible in nonsubstrates we concluded that a single conformation explains substrate specificities of DP IV. This conformation is characterized by the following dihedral angles: psi 1 = 85 degrees, omega 1 = 180 degrees, phi 2 = -75 degrees, psi 2 = 80 degrees, and omega 2 = 180 degrees. The conclusions were supported by comparisons of molecular electrostatic potentials calculated with the molecular graphics program HAMOG.


Changes in gamma-aminobutyric acid and somatostatin in epileptic cortex associated with low-grade gliomas.

The role of specific neuronal populations in epileptic foci was studied by comparing epileptic and non-epileptic cortex removed from patients with low-grade gliomas. Epileptic and nearby (within 1 to 2 cm) non-epileptic temporal lobe neocortex was identified using electrocorticography. Cortical specimens taken from four patients identified as epileptic and nonepileptic were all void of tumor infiltration. Somatostatin- and gamma-aminobutyric acid (GABAergic)-immunoreactive neurons were identified and counted. Although there was no significant difference in the overall cell count, the authors found a significant decrease in both somatostatin- and GABAergic-immunoreactive neurons (74% and 51%, respectively) in the epileptic cortex compared to that in nonepileptic cortex from the same patient. It is suggested that these findings demonstrate changes in neuronal subpopulations that may account for the onset and propagation of epileptiform activity in patients with low-grade gliomas.


Potential for hepatic and renal dysfunction during influenza B infection, convalescence, and after induction of secondary viremia.

Whether infection with influenza B virus alters hepatic function was examined in the ferret. Also, the possibility that viral-specific antibodies (Ab) could be produced well before their detection in serum was explored. During the febrile period of influenza, reductions in the serum potassium, anion gap, ammonia, albumin and CPK and elevations of the BUN, creatinine and the GGTP levels occurred. With convalescence, the electrolytes, BUN and creatinine normalized, FFA, SGPT and CPK levels rose and the serum GGTP rose even further. Hepatic fatty acid (FA) oxidation, ornithine transcarbamylase (OTC) and carnitine palmitoyltransferase (CPT) activities were minimally altered and liver ATP and total lipid content remained normal. Following experimental secondary viremia, serum FFA continued to rise, TG decreased and CPK remained elevated while SGPT and GGTP levels normalized. In the liver, FA oxidation and OTC rates remained unchanged but CPT activity was inhibited and the liver content of ATP was significantly reduced. Immune complex (IC) protein recovered from postmicrosomal supernatant fractions by polyethylene glycol precipitation was progressively increased in livers from convalescent and viremic animals. While the amount of IC protein recovered in the spleen also increases during convalescence, this is not the case after viremia when the IC formed seem to be processed largely by the liver. By SDS/PAGE, the major proteins identified in the IC were IgM and other viral proteins. However, the viral proteins could not be validated by immunoblot with Ab produced against purified influenza B hemagglutinin (HA) and neuraminidase (NA) most probably due to phagocytic alterations of glycoprotein immunodeterminants. These findings indicate that during influenza, convalescence and post viremia changes in the concentrations of several serum and liver components occur that reflect hepatic involvement. Also, antiviral Ab, largely IgM, appears to be produced early, complexes with Ag and can be found sequestered in both the liver and spleen at a time when Ab is not detectable in the serum.


Immunoreactivity for P-170 glycoprotein in malignant mesothelioma and in non-neoplastic mesothelium of the pleura using the murine monoclonal antibody JSB-1.

The results of an immunohistochemical study of P-170 glycoprotein immunoreactivity in human non-neoplastic mesothelium (35 cases) and in malignant mesothelioma (33 cases) using the murine monoclonal antibody JSB-1 are reported. The majority of malignant mesothelioma cases exhibited cytoplasmic and membrane immunoreactivity in neoplastic cells. These findings are highly significant when compared with the absence of immunoreactivity in normal mesothelium.


Effects of ursodeoxycholic acid on liver function in patients with cystic fibrosis and chronic cholestasis.

Ursodeoxycholic acid, 10 to 20 mg/kg per day, was administered for 1 year to 22 patients with cystic fibrosis and chronic cholestasis, resulting in significantly improved liver enzyme values. However, evidence of cholestasis continued, as shown by the pattern of alkaline phosphatase isoenzymes.


Benign recurrent cholestasis with normal gamma-glutamyl-transpeptidase activity.

We report two observations of intrahepatic cholestasis with normal serum levels of gamma-glutamyl-transpeptidase. These cases fit the diagnostic criteria of benign recurrent cholestasis and show that it, like Byler disease, is another form of pediatric intrahepatic cholestasis with a normal gamma-glutamyl-transpeptidase level in the infant.


Functional evidence for multiple gamma-aminobutyric acidB receptor subtypes in the rat cerebral cortex.

The aim of this work was the identification of pharmacologically distinct subtypes of gamma-aminobutyric acidB (GABAB) receptors in the central nervous system. Inasmuch as GABAB receptors are often sited on axon terminals where they mediate inhibition of transmitter release, we chose as models the GABAB receptors mediating inhibition of release of 1) endogenous GABA; 2) endogenous glutamate; and 3) somatostatin-like immunoreactivity (SRIF-LI). The experimental set up consisted of rat cerebrocortical synaptosomes depolarized in superfusion with 12 or 15 mM KCl. Endogenous GABA and glutamate were measured by high-performance liquid chromatography and SRIF-LI by radioimmunoassay. The selective GABAB receptor agonist (-)-baclofen inhibited in a concentration-dependent manner the K(+)-evoked release of GABA, glutamate and SRIF-Ll with similar potencies and efficacies [EC50 values, 1.1-1.5 microM; maximal inhibition, 45-50% at about 10 microM (-)-baclofen]. The GABAB receptor antagonist phaclofen concentration-dependently reduced the effects of (-)-baclofen on the release of GABA and SRIF-Ll but not on the release of glutamate, where it was ineffective up to 1000 microM. The rank order of potency (Ki values are shown in parentheses) are: SRIF-Ll (7.8 microM); GABA (10.4 microM); and glutamate (greater than 115 microM). The novel GABAB receptor antagonist 3-aminopropyl(diethoxymethyl) phosphinic acid (CGP 35348) displayed a different pattern on the three release systems examined (Ki values are shown in parentheses): SRIF-Ll (0.38 microM); glutamate (0.48 microM); and endogenous GABA (greater than 115 microM).(ABSTRACT TRUNCATED AT 250 WORDS)


Involvement of 5-hydroxytryptamine1A receptors in the modulation of micturition reflexes in the anesthetized rat.

Intravenous administration of the selective 5-hydroxytryptamine (5-HT)1A receptor agonist 8-hydroxy-2-(di-N-propylaminotetralin (8-OH-DPAT) and of a low doses of buspirone elicited the supraspinal micturition reflex (SMR) in urethane-anesthetized rats when the urinary bladder was filled with just a subthreshold volume of saline (threshold conditions). The effect of i.v. 8-OH-DPAT was abolished by hexamethonium or spiroxatrine. When SMR was elicited by bladder distension (suprathreshold conditions), i.v. 8-OH-DPAT increased the frequency of bladder contractions. In threshold conditions, stimulation of SMR was also induced by i.c.v. or by i.t. administration of 8-OH-DPAT and 5-HT but not by topical application of 8-OH-DPAT onto the bladder. Guanethidine pretreatment, which produced detrusor hyperreflexia, antagonized the effect of both i.c.v. and i.t. 8-OH-DPAT. In rats treated with capsaicin as adults, the response to 8-OH-DPAT was unchanged. In rats treated with capsaicin as newborns, instead, the response to i.t. 8-OH-DPAT was abolished and that to i.c.v. 8-OH-DPAT was shifted to higher doses. Pretreatment with 5,7-dihyroxytryptamine did not affect the response to i.t. 8-OH-DPAT but shifted to higher doses the response to i.c.v. 8-OH-DPAT. Intravenous administration of spiroxatrine, methysergide, NAN-190 [1-(2-methoxyphenyl)-4-[4-(2-phtalimido)butyl] piperazine] or high doses of buspirone but not of 1-sulpiride inhibited SMR in suprathreshold conditions. The inhibitory effect of spiroxatrine, NAN-190 and buspirone was not reduced by guanethidine pretreatment. In chronically spinalized animals, i.v. 8-OH-DPAT increased the amplitude of the reflex bladder contractions induced by bladder distension.(ABSTRACT TRUNCATED AT 250 WORDS)