Metabotropic glutamate receptors are required for the induction of long-term potentiation.

Recent observations have led to the suggestion that the metabotropic glutamate receptor may play a role in the induction or maintenance of long-term potentiation (LTP). However, experimental evidence supporting a role for this receptor in the induction of LTP is still inconclusive and controversial. Here we report that, in rat dorsolateral septal nucleus (DLSN) neurons, which have the highest density of metabotropic receptors and show functional responses, the induction of LTP is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate, but is blocked by two putative metabotropic glutamate receptor antagonists, L-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyrate. Furthermore, superfusion of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, a selective metabotropic glutamate agonist, resulted in a long-lasting potentiation of synaptic transmission similar to that induced by tetanic stimuli. Our results demonstrated that activation of postsynaptic metabotropic receptors is both necessary and sufficient for the induction of LTP in the DLSN, and we suggest that such a mechanism may be important at other CNS synapses.


The kinetics of the response to glutamate and kainate in neurons of the avian cochlear nucleus.

Neurons in the nucleus magnocellularis (nMAG) of the chicken precisely transmit auditory nerve activity via glutamatergic synapses. Using techniques for rapid application of solutions, we have explored the properties of CNQX-sensitive glutamate receptors in whole cells and outside-out patches from the nMAG. Glutamate-evoked current in patches desensitized biphasically to less than 1% of the peak current, with a fast time constant of 960 microseconds at 22 degrees C, decreasing to 570 microseconds at 33 degrees C. Dose-response studies using kainate indicated that at least two agonist molecules bind to gate the channel. We propose a kinetic model that quantitatively describes our experimental observations. The rapid kinetics of this receptor are well suited to allow phase locking of synaptic signals to auditory stimuli.


Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos.

The inner ear of all jawed vertebrates arises from the epithelium of the otic vesicle and contains three semicircular canals, otoliths, and sets of sensory neurons, all positioned precisely within the cranium to detect head orientation and movement. The msh-C gene and two new homebox genes, msh-D and a gene related to distal-less, dlx-3, are each expressed in distinct regions of the otic vesicle during its early development in zebrafish embryos. Cells in the ectoderm express dlx-3 before induction of the otic vesicle, suggesting that dlx-3 has an early function in this process. Later, cells aligned with the future axes of the semicircular canals specifically express either dlx-3 or msh-D. Even later, sensory hair cells express msh-C and msh-D, while other cells of the epithelium express dlx-3. The early expression of these genes could specify the orientation and morphogenesis of the inner ear, whereas their later expression could specify the fates of particular cell types.


Tissue-specific transcription of the rat tyrosine hydroxylase gene requires synergy between an AP-1 motif and an overlapping E box-containing dyad.

Transcription of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated in a tissue-specific manner. We have identified sequences from -205 to -182 as the minimal enhancer for TH in pheochromocytoma cells using site-directed mutagenesis. This segment (TGATTCAGAGGCAGGTGCCTGTGA) is composed of an AP-1 motif (TGATTCA) and an overlapping 20 bp dyad whose core resembles an E box site (CANNTG). Interaction between the two elements is necessary both in vivo and in vitro: mutation of either element caused a 65%-95% reduction in transcription, and the combination of the two elements conferred cell-specific activation on a heterologous promoter; separation of the two elements by an additional helical turn not only disrupted a DNA-protein complex unique to the two elements, but also abolished expression in vivo. Therefore, we conclude that the interaction between the AP-1 and the E box dyad motifs is responsible for cell-specific TH expression.


A new Conus peptide ligand for mammalian presynaptic Ca2+ channels.

Voltage-sensitive Ca2+ channels that control neurotransmitter release are blocked by omega-conotoxin (omega-CgTx) GVIA from the marine snail Conus geographus, the most widely used inhibitor of neurotransmitter release. However, many mammalian synapses are omega-CgTx-GVIA insensitive. We describe a new Conus peptide, omega-CgTx-MVIIC, that is an effective inhibitor of omega-CgTx-GVIA-resistant synaptic transmission. Ca2+ channel targets that are inhibited by omega-CgTx-MVIIC but not by omega-CgTx-GVIA include those mediating depolarization-induced 45Ca2+ uptake in rat synaptosome preparations, "P" currents in cerebellar Purkinje cells, and a subset of omega-CgTx-GVIA-resistant currents in CA1 hippocampal pyramidal cells. The characterization of omega-CgTx-MVIIC by a combination of molecular genetics and chemical synthesis defines a general approach for obtaining ligands with novel receptor subtype specificity from Conus.


Sympathetic neurons mediate developmental change in cardiac sodium channel gating through long-term neurotransmitter action.

Innervation of nerve and muscle cells during development is often accompanied by changes in the expression and function of ion channels in the postsynaptic cell. However, the signaling pathways whereby the presynaptic nerve influences the properties of the postsynaptic cell are less well understood. Indirect evidence suggests that cardiac voltage-gated Na+ channels undergo important changes during development. Here, we compare directly single voltage-gated Na+ channel currents from neonatal and adult rat ventricular myocytes and report a negative shift in the voltage dependence of channel gating during development, leading to a significant speeding of channel activation and inactivation at a fixed membrane potential. These developmental changes can be mimicked in vitro by innervation of neonatal myocytes with sympathetic neurons. The effect of sympathetic neurons is blocked by the beta-adrenergic receptor antagonist propranolol and is mimicked by prolonged coculture of neonatal myocytes with a membrane-permeable cAMP analog. Thus presynaptic neurons can control the developmental phenotype of ion channels in a postsynaptic cell through a classic receptor-mediated neurotransmitter action that involves a defined second messenger pathway.


Impairment of carbamazepine-10, 11-epoxide elimination by valnoctamide, a valpromide isomer, in healthy subjects.

The effect of the valpromide isomer valnoctamide (VCD, 200 mg three times daily for 8 days), an over-the-counter tranquillizer, on the elimination kinetics of a single oral dose of carbamazepine-10, 11-epoxide (CBZ-E, 100 mg) was investigated in healthy subjects. During VCD treatment, the half-life of CBZ-E was prolonged significantly compared with control (19.7 +/- 6.7 h vs 6.9 +/- 2.0 h, means +/- s.d., P less than 0.01), and its oral clearance decreased four-fold (from 109.6 +/- 30.7 to 28.8 +/- 11.1 ml h-1 kg-1, P less than 0.01). These findings indicate that VCM, like valpromide, strongly inhibits epoxide hydrolase in vivo.


Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.


Modulation of P-glycoprotein phosphorylation and drug transport by sodium butyrate.

P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents. Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased. In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux. Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function. This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp. Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment. Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation. These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.


Proteolytic processing of human lysosomal arylsulfatase A.

Arylsulfatase A purified from human placenta contained an unreported component with an apparent molecular mass of 7 kDa in addition to the two known components with apparent molecular masses of 58 and 50 kDa. The detailed relationship between the 58 kDa component and the 50 kDa component is as yet unknown. The present study was undertaken to define the structure of the subunits of the sulfatase. The N-terminal sequence of the 50 kDa component was identical to that of the 58 kDa component. Furthermore, the peptide maps of the 50 kDa component, which was separately digested with trypsin and Achromobacter proteinase I, were quite similar to those of the 58 kDa one. Through sequence analysis of the incompatible peaks in the peptide maps, the 50 kDa component was found to lack a sequence from Val-445 to the C-terminus. On the other hand, the N-terminal sequence of the 7 kDa component began with Ala-448, though there was a minor sequence commencing with Thr-449. These observations suggest that the 50 and 7 kDa components were produced by limited proteolysis near the C-terminus of the 58 kDa component. Through analysis using unreducing SDS-PAGE, the 58 and the 7 kDa components were found to be linked by disulphide bonds. Arylsulfatase A purified from human liver was also composed of the same subunits as the placental one. This finding suggests that human arylsulfatase A undergoes similar proteolytic processing regardless of the tissue involved.


MK-801 selectively protects mouse arcuate neurons in vivo against glutamate toxicity.

The receptor mediating glutamate toxicity in vivo has not yet been identified. The effects of NMDA antagonist MK-801 and the AMPA antagonist NBQX were studied on glutamate toxicity in the arcuate nucleus of newborn and adult mice. Morphometric methods were used to determine the effects of antagonists on glutamate toxicity. In the developing arcuate, MK-801 abolished glutamate neurotoxicity whereas NBQX has no effect. MK-801, but not NBQX, afforded significant but not complete protection in mature arcuate neurons. The residual toxicity could not be blocked by co-administration of NBQX which paradoxically partially inhibited the protective effect of MK-801. The results show that throughout development, glutamate neurotoxicity in the arcuate nucleus is predominantly mediated by NMDA receptors.


Glutamate receptors mediate acoustic input to the reticular brain stem.

Previous studies have shown that many neurons of the pontine reticular brain stem respond to acoustic stimulation. However, it was not clear which neurotransmitter is involved in the mediation of auditory information. As glutamate appears to be a prominent transmitter in the auditory system, we iontophoretically applied antagonists of the AMPA/kainate- and NMDA-receptors to reticular neurons. Both glutamate antagonists reduced the acoustically evoked response, with the AMPA/kainate-receptor antagonist being more efficient. As the neurons showed a short latency and a high intensity threshold to the acoustic stimuli and most of them appeared to project into the spinal cord, we conclude that glutamate receptors on reticulospinal pontine brain stem neurons probably mediate auditory short-latency behaviour, such as the startle response.


Nucleotide sequence of a gene from Phanerochaete chrysosporium that shows homology to the facA gene of Aspergillus nidulans.

Heterologous hybridisation was used to isolate a genomic DNA sequence from Phanerochaete chrysosporium using the facA (acetyl CoA synthetase) gene from Aspergillus nidulans as a probe. The cloned sequence hybridises to a 2.2 kb transcript in poly(A)+ RNA prepared from mycelium grown on acetate as the sole carbon source. Comparison of the DNA sequence obtained with those of the A. nidulans facA and N. crassa acu5 genes reveals an ORF that appears to be interrupted by five typical fungal introns. Two possible candidates for the translation initiation codon were observed. Homology with the facA and acu5 genes is revealed after the second ATG codon.


Bronchodilator-resistive cough in atopic patients: bronchial reversibility and hyperresponsiveness.

The number of atopic patients presenting only chronic non-productive cough appears to be increasing. This study was conducted to confirm the existence of non-asthmatic cough associated with atopy. We prospectively examined atopic findings, therapeutic effects of inhaled procaterol, azelastin, and/or glucocorticoids, improvement of FEV1 by bronchodilator therapy and bronchial responsiveness to methacholine in 20 patients. The cough was relieved by inhaled procaterol in 10 patients (Group 2) but not in the other 10 patients (Group 1). The increase in FEV1 by inhaled salbutamol following aminophylline injection was significantly less in Group 1 than in Group 2. Bronchial responsiveness to methacholine was normal in Group 1 while that in Group 2 was hyperreactive. These findings indicate that there is atopic non-asthmatic bronchodilator-resistive cough (Group 1) which is a different entity from bronchodilator-responsive cough (Group 2), or the so-called "cough variant asthma".


Unilateral facial swelling and exophthalmos in a patient with polyarteritis nodosa.

A patient with polyarteritis nodosa (PN) presenting with exophthalmos and facial swelling, which are rarely found in PN, is reported. The patient, a 27-year-old male, complained of painful facial swelling and diplopia. Physical examinations showed facial swelling around the right orbit and exophthalmos. After admission, he experienced myalgia in both calves. Laboratory studies disclosed leukocytosis and liver dysfunction. Celiac and renal angiograms showed aneurysms. A biopsy specimen of his left calf showed an arterial inflammatory process. The patient was diagnosed as having PN. He had an excellent response to corticosteroids, with prompt improvement.


CSF corticotropin-releasing hormone and somatostatin in major depression: response to antidepressant treatment and relapse.

Immunoreactive corticotropin-releasing hormone (CRH) and somatostatin (SRIF) were measured in the cerebrospinal fluid (CSF) of 24 female in-patients, suffering from DSM-III-R major depression, both before and after antidepressant treatment. In the total group there were no significant differences between pre- and post-treatment CSF-CRH and SRIF concentrations despite satisfactory clinical improvement in each patient. However, there was a significant post-treatment reduction of the CSF-CRH concentration in the 15 patients who remained depression-free for at least 6 months following treatment, in contrast to the tendency for elevation in those 9 subjects who relapsed within 6 months. CSF-SRIF showed no similar pattern. High, or even increasing, CSF-CRH concentration during antidepressant treatment may indicate lack of normalization of an underlying process in major depression despite symptomatic improvement and predicted early relapse.


Additive effects of chronic treatment with antidepressant drugs and intermittent treatment with a dopamine agonist.

Locomotor activity in response to the D2/D3 agonist quinpirole was studied in male rats treated chronically (25-40 days) with imipramine, amitriptyline or mianserin (5 mg/kg/day). All three antidepressants potentiated the response to quinpirole. Repeated administration of quinpirole at 3-day intervals also caused a marked increase in the locomotor response to quinpirole. Imipramine-treated animals were more active than vehicle-treated animals on all quinpirole trials. Animals tested in the locomotor activity apparatus for the first time following their fifth quinpirole injection showed sensitization, but to a lesser extent than animals tested repeatedly under quinpirole. This context-independent sensitization was potentiated by all three antidepressants. We discuss the potential clinical relevance of these results.


Sigma receptor modulation of the muscle relaxant action of eperisone.

We examined the mechanism of the muscle relaxant action of eperisone, using Straub tail and binding studies. In vivo, eperisone (50 and 100 mg/kg i.p.) produced a dose-dependent inhibition of the Straub tail in mice and this inhibitory effect was significantly reversed by haloperidol (0.5 mg/kg i.p.). This drug in itself had no effect on the Straub tail. Sulpiride (50 mg/kg i.p.) failed to reverse the inhibitory effect of eperisone. In vitro, (+)-3-(3-[3H]hydroxyphenyl)-N-(1-propylpiperidine) ((+)-[3H]3-PPP) specific binding, in rat brain membrane, was prevented by eperisone and the IC50 value was 0.43 nM. The muscle relaxant action of eperisone may be modulated by sigma receptors.


Nonoperative treatment of neurosurgical infections.

The indications for nonsurgical management of CNS infections manifested by collections of pus are very limited. In general, such management is restricted to neurologically intact patients who are unable to undergo the needed surgical procedure and in whom the organism can be identified presumptively from other cultures. In such circumstances, brain abscesses less than 1.5 cm in diameter, small subdural empyemas, some spinal epidural abscesses, and many cases of spinal osteomyelitis can be successfully treated with prolonged intravenous antibiotic therapy. In patients who are unable to tolerate a neurosurgical procedure, the presence of an infectious CNS mass lesion is not hopeless and vigorous medical therapy offers the possibility of cure.


Mouse F9 teratocarcinoma stem cells expressing the stably transfected homeobox gene Hox 1.6 exhibit an altered morphology.

Expression of the murine homeobox gene Hox 1.6 rapidly increases in F9 teratocarcinoma cells when these cells are induced with retinoic acid to differentiate into primitive and parietal endoderm. Hox 1.6 encodes a putative transcriptional regulatory protein which may function as a secondary regulator of gene expression during the differentiation process. To examine the role of the Hox 1.6 gene, we have stably transfected F9 stem cells with a cDNA containing the complete coding sequence of Hox 1.6 under the control of the mouse metallothionein promoter. Two clonally distinct cell lines that express high levels of the transfected Hox 1.6 gene have been isolated and characterized. We show that expression of the transfected Hox 1.6 gene in F9 cells dramatically alters the stem cell morphology. However, the transfected cells do not differentiate in the absence of retinoic acid treatment, nor are they prevented from differentiating in response to such treatments. We therefore suggest that the Hox 1.6 gene controls the expression of genes which influence changes in F9 cell morphology during RA-induced differentiation.


Infections of the dural spaces.

Suppuration involving the epidural and subdural spaces is a rare occurrence in modern neurosurgical practice. Early diagnosis and appropriate treatment of infections of the dural spaces may avert the high incidence of neurologic disability and death traditionally associated with them, however. Prompt neurosurgical intervention in the treatment of these lesions has been the standard with which all other therapies have been compared.


Peripheral blood stem cell transplantation.

Haematopoietic stem cells are usually sessile within the bone marrow microenvironment. However, small numbers do circulate in the peripheral blood of normal individuals, and following chemotherapy and/or intravenous growth factors, a substantial transient rise in circulating stem cells occurs. Leukocytes harvested by cytapheresis at this time can be used for autologous reconstitution of the haematopoietic and lymphoid systems following high dosage chemo/radiotherapy for the treatment of malignant disease. Peripheral blood stem cell transplants give rise to similar disease response rates as autologous bone marrow transplants, but have the advantage of more rapid haematopoietic reconstitution, and in addition can be offered to patients in whom marrow harvest is not feasible due to bone marrow damage or infiltration. This article reviews the theoretical and historical background to haematopoietic stem cell research, current clinical practice in peripheral blood stem cell mobilisation and harvesting, addresses the potential advantages and disadvantages compared to bone marrow transplantation, and assesses current experience of comparative efficacy.


A microcomputer FORTRAN program for rapid determination of lethal concentrations of biocides in mosquito control.

Probit analysis calculations are highly useful in biology and related sciences. Since the statistical calculations and tests required are quite involved, the use of an automatic computer program is desirable. The description of the computational procedures and use of the computer program with suitable examples from mosquito control programmes are discussed.


Homozygosity for the transthyretin-Met30-gene in seven individuals with familial amyloidosis with polyneuropathy detected by restriction enzyme analysis of amplified genomic DNA sequences.

Familial amyloidotic polyneuropathy (FAP) with a mutation in position 30 of transthyretin (TTR) (previously called prealbumin) is an autosomal dominant inherited disorder characterized by varying degrees of peripheral neuropathy, nephropathy, gastrointestinal problems, and vitreous amyloid. We have earlier diagnosed homozygosity for the TTR-Met30-gene using Southern analysis in four Swedish individuals. We have found it possible to detect homozygosity for the Met-30 mutation by amplifying discrete regions of the TTR-gene using polymerase chain reaction (PCR), and the amplification products restricted with NsiI analysed by gel electrophoresis. Clinical data on seven homozygous individuals, including three new cases, are presented.


Cranial nerve involvement in childhood polyarteritis nodosa.

Amongst a variety of neurological manifestations of childhood polyarteritis nodosa, cranial nerve involvement is unusual. We report 4 cases with cranial nerve palsies in a series of 36 biopsy-proven patients. Two cases presented with IIIrd nerve palsy alone, one with right IIIrd and left IVth nerve palsy, and one with peripheral VIIth nerve paresis. All 4 patients showed good response to prednisolone and cyclophosphamide treatment. Cranial nerve involvement in childhood polyarteritis nodosa seems not so rare when patients are followed on long term basis.


Chronic treatment with L-threonine in amyotrophic lateral sclerosis: a pilot study.

Thirty patients suffering from amyotrophic lateral sclerosis were included in an open therapeutical trial. They were randomized to receive either L-threonine (Thr), a precursor of the inhibitory amino acid glycine, or vitamin B or carnitine. Thirteen patients (9 patients on Thr and 4 control subjects) completed the 1-year trial. No statistical differences were observed between the treated group and the control patients in the decline of the clinical assessment score. Nevertheless, Thr-treated patients complained less frequently of respiratory failure than the control group despite bulbar involvement being more common in the Thr group at entry.


Reconstruction of the burned foot.

Burns of the feet pose unique and difficult problems in initial management, reconstruction, and the attainment of long-term functional results. The primary reconstructive goals for this region are unimpeded ambulation and weightbearing. These objectives can be achieved by adherence to established principles of wound management, a clear delineation of the reconstructive requirements of the foot, and a team approach toward attaining these goals.


Zuclopenthixol, melperone and haloperidol/levomepromazine in the elderly. Meta-analysis of two double-blind trials at 15 nursing homes in Norway.

A meta-analysis was carried out of the data from two double-blind, multi-centre studies with identical methodology which compared the effectiveness of treatment of elderly patients with zuclopenthixol and with other antipsychotic drugs. In one study, patients were treated for 4 weeks with either zuclopenthixol or melperone; in the other, with either zuclopenthixol or a combination of haloperidol and levomepromazine. The meta-analysis evaluated the results of 96 patients, 49 in the zuclopenthixol group and 47 in the comparison group. Doses, which were adjusted to individual patient's needs, were low as shown by the percentages of defined daily doses for each of the study drugs. The results indicated that all three treatment alternatives were effective in the treatment of elderly patients with symptoms of agitation and hostility/aggressiveness. There was a trend, however, for zuclopenthixol to have a more rapid onset of effect. Zuclopenthixol also has the advantage for both patients and nursing staff that dosage is once daily. Only few and mild side-effects were reported with the three drug regimens.


Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki-67 antibody.

This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki-67 antibody, or the proliferation-associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL-60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra- and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki-67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p120 was not significantly affected by 0.65-1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki-67.(ABSTRACT TRUNCATED AT 250 WORDS)


The intravenous self-administration of antihistamines by rhesus monkeys.

Rhesus monkeys were trained to lever press for infusions of cocaine during daily, 1-h experimental sessions. Following stabilization of the cocaine-maintained baselines, various antihistamines were substituted for cocaine to determine whether they would be self-administered. The results indicated that all monkeys tested self-administered tripelennamine and chlorpheniramine. One monkey out of the four self-administered pyrilamine, but only at a single (300 microgram/kg) high dose. Phenyltoloxamine, cimetidine and hydroxyzine were not self-administered. These results further illuminate differences amongst H1 antagonists in their potential for self-administration and, when examined in context with other reports, suggest that stimulant-like properties may help mediate their reinforcing effects when present.


Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.


A TaqI polymorphism in the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro.

In the present study we searched for restriction fragment length polymorphisms (RFLP) in the human interleukin-1 beta (IL-1 beta) gene and for correlations to monocyte (Mo) function in non-related healthy donors and insulin-dependent diabetic patients. We demonstrated a diallelic polymorphism with the restriction enzyme TaqI consisting of fragments of 9.4 kb and 13.4 kb. No differences in allele or genotype frequencies of this RFLP were observed between randomly selected controls and randomly selected patients with insulin-dependent diabetes mellitus (IDDM). However, when analysing IDDM patients negative for HLA-DR3 and -DR4, our data demonstrate that the 13.4 kb allele is more frequent in this group compared to a matched control group. The functional impact of this RFLP was studied by analysing in vitro stimulated Mo IL-1 beta response. An IL-1 beta allele dosage effect on secretory capacity was observed after LPS-stimulation: 13.4/13.4 kb homozygous individuals secreted significantly more IL-1 beta than 9.4/13.4 kb heterozygous individuals, who secreted significantly more than 9.4/9.4 kb homozygous individuals. Analyses of supernatants from LPS-stimulated Mo cultures from individuals with each TaqI IL-1 beta genotype revealed no differences in the mouse thymocyte co-stimulatory assay when compared on a molar basis, indicating that the TaqI polymorphism gave rise only to quantitative differences in expression levels and probably not to a mutant IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanism of catalysis of Fe(II) oxidation by ferritin H chains.

Recombinant H chain ferritins bearing site-directed amino acid substitutions at their ferroxidase centres have been used to study the mechanism of catalysis of Fe(II) oxidation by this protein. UV-difference spectra have been obtained at various times after the aerobic addition of Fe(II) to the recombinants. These indicate that the first product of Fe(II) oxidation by wild type H chain apoferritin is an Fe(III) mu-oxo-bridged dimer. This suggests that fast oxidation is achieved by 2-electron transfer from two Fe(II) to dioxygen. Modelling of Fe(III) dimer binding to human H chain apoferritin shows a solvent-accessible site, which resembles that of ribonucleotide reductase in its ligands. Substitution of these ligands by other amino acids usually prevents dimer formation and leads to greatly reduced Fe(II) oxidation rates.


Nucleotide sequence of a cDNA encoding human tumor necrosis factor beta from B lymphoblastoid cell RPMI 1788.

Published sequences of cDNA for human tumor necrosis factor beta (TNF-beta) have a discrepancy within the coding region as well as exon 1. To resolve these discrepancies we have re-isolated TNF-beta cDNA from the human B cell lymphoblastoid cell line, RPMI 1788, and determined its DNA sequence. Results indicate that amino acid 26 is threonine (Thr) instead of asparagine (Asn). In contrast to published sequences, the sequence of the exon 1 region corresponded to the genomic sequence of TNF-beta. From our studies we conclude that the TNF-beta gene of the human B cell lymphoblastoid cell line, RPMI 1788, is homologous with respect to the TNF-beta gene.


Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.


Characterization of the pcp gene encoding the pyrrolidone carboxyl peptidase of Bacillus subtilis.

Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp) is an enzyme that catalyzes the removal of the N-terminal pyroglutamyl group from some peptides or proteins. Its value in protein chemistry and bacterial diagnosis makes this enzyme an interesting subject of study. The present paper reports for the first time the cloning and characterization of a pyrrolidone carboxyl peptidase gene (pcp). This gene is present in a single copy in the genome of Bacillus subtilis as indicated by Southern blot hybridization analysis. The pcp transcripts were analyzed in Escherichia coli by Northern blot hybridization and S1 nuclease mapping. The deduced amino acid sequence predicts a protein of 215 amino acids with a calculated molecular weight of 23,777 Da. The pcp gene has been over-expressed in E. coli, allowing the identification and partial characterization of Pcp protein.


Computational approach towards the three-dimensional structure of E. coli tyrosine aminotransferase.

We present a new model for E. coli tyrosine aminotransferase based on the X-ray structures of the wild type and Val39Leu mutant of E. coli aspartate aminotransferase and computer simulation studies. Active site characteristics of the model are correlated with experimental observations on the specificity of these enzymes towards aromatic/dicarboxylic acid substrates.


The effect of two dopaminergic drugs on menstrual function and psychological state in hyperprolactinemia.

To investigate the effect of dopaminergic drugs on the well being in hyperprolactinemic patients. A psychometric test for well being, the SCL-90, was applied at baseline and in the 24th week of a double-blind randomized prospective study comparing the effectiveness and safety of the new dopamine d2 agonist CV 205-502 with bromocriptine. Outpatient department of a university clinic for obstetrics and gynecology. Twenty-four women with hyperprolactinemia, 9 of whom had a prolactinoma. Twenty had been treated before, and 11 were known to react unfavorably to bromocriptine. The effectiveness of CV 205-502 was identical to bromocriptine: its tolerability appeared to be better, especially in the initial phase of treatment. At baseline, the mean scores of the SCL-90 were significantly elevated over the reference values. Sixteen patients had normal scores. The elevations were caused by 8 patients with scores in the range found in psychiatric disease (211 +/- 30 [SD] [CV 205-502] and 182 +/- 32 [bromocriptine]). They were depressed, anxious, and hostile. At 24 weeks, the patients treated with CV 205-502 scored better (130 +/- 23.5) in the SCL-90 than the patients treated with bromocriptine (149.5 +/- 20). The markedly increased well being in patients treated with CV 205-502 cannot be explained by its better tolerability and is probably caused by a specific central activity of CV 205-502. Further research into the antidepressive properties of this compound is warranted.


[The TSH receptor gene and the pathogenesis of Graves' disease].

The molecular cloning of the thyrotropin (TSH) receptor cDNA has led to advances in understanding the structure and function of the molecule and the pathogenesis of autoimmune thyroid disease. The recombinant TSH receptors expressed on mammalian cells are now available for TSAb and TBIAb assays. TBI assay in this system is much more sensitive than the conventional method. The binding sites for TSH and TSAb/TBIAb have been studied with epitope library, synthetic peptides, anti-peptide sera and mutagenesis. Some of these data, however, are confusing and undefined. The binding sites for TSH and TSAb/TBIAb are very likely to span the entire region of the extracellular domain of the TSH receptor with discontinuous contact points, and seem to be different from each other on N-terminal half, but similar on C-terminal half, of the extracellular domain of the receptor. However, the determination of the precise amino acids involved may be very difficult without monoclonal anti-TSH receptor antibodies.


Molecular strategies in genetic diagnosis of transthyretin-related hereditary amyloidosis.

Transthyretin-related hereditary (TTR) amyloidoses represent a clinically heterogeneous group of diseases associated with various point mutations of the TTR gene. We propose a molecular strategy for the diagnosis of this group of disorders.


Expression of drug-metabolizing enzymes and P-170 glycoprotein in colorectal carcinoma and normal mucosa.

Resistance to chemotherapy is a significant problem in the treatment of colorectal carcinomas. To obtain insight into the mechanism of drug resistance, the expression of P-170 glycoprotein and biotransformation enzymes that are potentially able to contribute to drug resistance were investigated in paired samples of normal mucosa and tumors from 24 patients with colorectal cancer. In the tumors, glutathione S-transferase (GST) enzyme activity and content of GST-pi and P-170 glycoprotein were increased significantly compared with normal mucosa (P less than 0.03, P less than 0.003, and P less than 0.02, respectively). In contrast, GST-alpha and -mu, present in minor amounts compared with GST-pi, were downregulated in the tumor. Cytochrome P-450(4,5,6) and UDP-glucuronyltransferase (towards 4-nitrophenol and bilirubin) levels were significantly lower in the tumors (P less than 0.0001 and P less than 0.0002, respectively). Because decreased expression of cytochrome P-450 and increased levels of GST-pi and the P-170 glycoprotein have been implicated in (multi)drug resistance, these findings strongly suggest that in colorectal tumors the inherent resistance is multifactorial. Research to overcome this resistance should therefore be directed toward a combined treatment that eliminates all of these different mechanisms.


Mucin and protein release in the rabbit jejunum: effects of bethanechol and vagal nerve stimulation.

The role of the vagus nerve and cholinergic mechanisms in the control of rabbit jejunal mucin and protein release was investigated in vivo. In anesthetized animals, a 10-cm segment of the jejunum was cannulated and perfused with saline. Perfusate was collected and analyzed for mucin (by immunoassay) and protein. Bilateral cervical vagotomy had no effect on basal mucin or protein output, suggesting that the vagus nerve does not exert a tonic control on jejunal macromolecule secretion. Electrical stimulation of the vagi did not alter mucin release, even in the presence of muscarinic cholinergic (scopolamine) or adrenergic (propranolol and phentolamine) blockade. In contrast, protein output increased significantly after vagal stimulation, an effect inhibited by scopolamine. In both vagotomized and vagally intact rabbits, the cholinergic agonist bethanechol (200 micrograms/kg intraperitoneally) induced a scopolamine-sensitive increase in both mucin and protein output. Predominantly serum proteins were released into intestinal perfusates after vagal or cholinergic stimulation. It is concluded that the extrinsic vagus nerve does not regulate rabbit jejunal mucin secretion in vivo and that cholinergic control of intestinal goblet cells is implemented entirely by the intrinsic enteric nervous system. In addition, cholinergic or vagal stimulation increases intestinal vascular and epithelial permeability, resulting in the passage of serum proteins into the lumen, possibly by opening tight junctions and paracellular pathways.


Apolipoprotein B-gene DNA polymorphisms (XbaI and EcoRI), serum lipids, and apolipoproteins in healthy Chinese.

The frequency of restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene, detected by XbaI and EcoRI, and their influence on serum lipids and apolipoproteins were studied in healthy Chinese of both sexes in Singapore. A total of 221 subjects (150 males, 71 females) were investigated for the XbaI and 159 subjects for the EcoRI polymorphisms, while serum lipids and apolipoprotein levels were available for 196 subjects. The frequency of the X2 allele was found to be significantly lower in the Chinese than that reported in Caucasians from the United Kingdom (0.09 vs. 0.51, P less than 0.001). The haplotype frequencies were also significantly different between the Chinese and Caucasians with a higher frequency of X1R1 in the former compared to the latter (0.85 vs. 0.34, P less than 0.0001). The distribution of RFLP genotypes at both of the restriction sites was at Hardy-Weinberg equilibrium in all groups. The influence of the apo B RFLPs on serum lipids and apolipoprotein levels (apo AI, AII, and B) was studied by both residual and multiple regression analyses considering age, sex, body mass index (BMI), and genotypes as independent variables in all possible combinations. No association was observed between the apo B genotypes and serum lipids or apolipoprotein levels except for high density lipoprotein cholesterol (HDLC), apo AI and AII, with the X2 being associated with significantly lower levels of HDLC as well as apo AI and AII, the effect being stronger in males. These data raise the possibility that the mechanism of reported association between apo B polymorphism and coronary artery disease may be through effects on HDLC.


DNA-binding activity of the murine homeodomain protein Hox-2.3 produced by a hybrid phage T7/vaccinia virus system.

Homeobox-containing genes encode transcription factors that, via the homeodomain, bind specifically to DNA. To study the DNA-binding properties of the murine homeodomain-containing protein, Hox-2.3, a hybrid expression system was used, combining gene expression by recombinant vaccinia virus (reVV) with bacteriophage T7 transcription. Expression was achieved by co-infecting HeLa cells with two reVVs, one expressing the T7-RNA polymerase-encoding gene directed by the VV promoter, P7.5, and another containing the Hox-2.3 coding sequence under control of a T7 promoter [Fuerst et al., Mol. Cell. Biol. 7 (1987) 2538-2544]. Co-infected HeLa cells produced large amounts of full-length Hox-2.3 protein. Cytoplasmic and nuclear extracts from these cells were used to examine DNA-binding specificity in vitro. reVV-produced Hox-2.3 protein bound to oligos that contained one or several copies of the common homeodomain-binding site, 5'-TCA-ATTAAAT, and to a lesser extent to multiple (TAA) repeats. Using Southwestern blot analysis, no Hox-2.3-binding sites were detected in a region of the Hox-2 cluster containing the Hox-2.3, Hox-2.4 and Hox-2.5 genes.


A Xenopus borealis homeobox gene expressed preferentially in posterior ectoderm.

We have isolated a new homeobox-encoding gene (XhoxB.1) from Xenopus borealis which has a homeobox exon identical to that of the murine Hox1.7 gene, except for one amino acid. XhoxB.1 transcripts of 2.1 and 7.0 kb are first detected at the late gastrula stage, accumulate until the tailbud stage and are most abundant in the posterior third of the dorsal part of the embryo.


Complications of peptic ulcer disease before and after the introduction of H2-receptor antagonists.

This study was undertaken in order to evaluate the incidence of operations for bleeding, perforated and obstructing peptic ulcers in a defined population before and after the introduction of H2-receptor antagonists. The annual incidence of surgery for all peptic ulcer complications increased slightly, from 6.9 per 10(5) individuals in 1977 to 14.2 per 10(5) in 1989 (n.s.), whereas the annual incidence of operations for ulcer bleeding and perforation remained relatively stable, varying from 2.8 to 8.9 per 10(5) inhabitants and from 2.3 to 7.5 per 10(5) inhabitants during the study period. Operations performed for gastric outlet obstruction did not increase, varying from 0.8 to 2.2 per 10(5) individuals over the study period. The annual proportion of emergency operations did not increase. Young men and old women were often operated on for bleeding (p less than 0.0001) and perforated ulcers (p less than 0.01). Duodenal ulcer bleeding and perforation were more frequent in the young patient groups. Overall mortality after operations performed for bleeding was 15%, and that after operations for perforation or obstruction, 17% and 8%, respectively. The mean age of the fatalities, 63 +/- 13 years, was significantly higher than that of those who survived after operation, 53 +/- 15 years (p 0.0001). Mortality was higher after operations for gastric ulcer complications (22%) than after operations for duodenal ulcer complications (10%) (p less than 0.01).


Complications associated with total parenteral nutrition in infants with short bowel syndrome.

The use of total parenteral nutrition (TPN) in five out of six infants with short bowel syndrome (SBS) adaptation permitted enteral nutrition. The duration of TPN depended on the extent of the resection, whether it was proximal or distal, and the adaptation of the residual gut. Residual bowel measuring 10 cm required prolonged TPN in the sixth infant and was not compatible with survival. Catheter-related complications were infection, malposition and dislodgement of the catheter. Metabolic complications were easily controlled by regulating the concentration of the infusate and the rate of the infusion. Osteopenia was common in prolonged TPN and was corrected with vitamin D supplementation. Cholestasis was the most common complication. It was demonstrated with elevation of gamma-glutamyl-transpeptidase levels which became evident as early as six weeks after the introduction of TPN. Serum bilirubin and transaminase elevations were later manifestations. One infant died of hepatic decompensation. Late morphological manifestations were those of cholestatic changes with fibrosis. The biochemical abnormalities of cholestasis were reversible provided TPN was discontinued at an early stage.


Effects of exogenous somatostatin and insulin on islet amyloid polypeptide (amylin) release from perfused rat pancreas.

This study examined the effects of exogenous somatostatin and insulin on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas. Somatostatin inhibited the release of both amylin and insulin from the perfused pancreas to the same extent. The infusion of 10 nM somatostatin resulted in 40% inhibition of the secretion of both amylin and insulin induced by 11.1 mM glucose and 10 mM arginine, and this inhibition was significantly increased to 70% by the infusion of 100 nM somatostatin (p less than 0.05). The amylin/insulin molar ratios remained constant at 0.8% and were not changed by the infusion of somatostatin. On the other hand exogenous insulin at a concentration of 1.8 nM did not affect the release of amylin induced by 11.1 mM glucose and 10 mM arginine, whereas 180 nM insulin slightly, although not significantly, inhibited the release of amylin by 15%. These findings suggest that the release of amylin may be negatively regulated by somatostatin and that circulating insulin may have no direct effect on the release of amylin at least at a physiological concentration.


A new polymorphic site in the G6PD gene.

A polymorphic restriction site has been found in intron 11 of the gene for glucose-6-phosphate dehydrogenase (G6PD). This site is produced by a T----C substitution 13 bp upstream of exon 12, producing an NlaIII restriction site. In various populations there was a strong association between a T at nt 1311 of the G6PD cDNA and the presence of the NlaIII restriction site. Among African Americans, however, the presence of a C at nt 1311 was sometimes associated with the presence of a polymorphic NlaIII site.


Exclusion of stromelysin-1, stromelysin-2, interstitial collagenase and fibronectin genes as the mutant loci in a family with recessive epidermolysis bullosa dystrophica and a form of cerebellar ataxia.

The interstitial collagenase gene (CLG), one of the main candidates in severe generalized recessive epidermolysis bullosa dystrophica (SGREBD), is closely linked to the stromelysin-1 (STMY1) and stromelysin-2 (STMY2) genes. These three loci map on chromosome 11 (q21-q22.3), where they constitute a cluster of genes coding for metalloproteinases involved in the degradation of the extracellular matrix (ECM). A recessive form of cerebellar ataxia of post-puberal onset (CLA1) has also been assigned to chromosome 11 (q14-q21). Since useful restriction fragment length polymorphisms (RFLPs) for the CLG gene are not available, we have studied the inheritance of the marker TaqI RFLP of the STMY1 gene in a North Italian family with a child affected by SGREBD, and his two sisters showing cerebellar ataxia (CA) of post-puberal onset. We have also studied the MspI RFLP of the fibronectin gene (FN1), which is located on chromosome 2q34-q36, and which codes for non-collagenous matrix proteins. Since we did not observe the segregation of the pathological phenotypes with STMY1 and FN1 RFLPs, we excluded the involvement of these genes in both the SGREBD and CA present in this family. The exclusion of the STMY1 gene indicates that the mutation causing SGREBD cannot be located in the CLG and/or STMY2 genes because of their proximity to the STMY1 locus. These data also indicate that the CA form here reported is not attributable to alterations in regions close to the collagenase cluster on chromosome 11.


Reduced recombination and paternal age effect in Klinefelter syndrome.

The parental origin of the additional sex chromosome was studied in 47 cases with an XXY sex chromosome constitution. In 23 cases (49%), the error occurred during the first paternal meiotic division. Maternal origin of the additional chromosome was found in the remaining 24 cases (51%). Centromeric homo- versus heterozygosity could be determined in 18 out of the 24 maternally derived cases. According to the centromeric status and recombination rate, the nondisjunction was attributable in 9 cases (50%) to an error at the first maternal meiotic division, in 7 cases (39%) to an error at the second maternal meiotic division and in 2 cases (11%) to a nullo-chiasmata nondisjunction at meiosis II or to postzygotic mitotic error. No recombination, and in particular none in the pericentromeric region, was found in any of the 9 cases due to nondisjunction at the first maternal meiotic division. Significantly increased paternal age was found in the paternally derived cases. Maternal age was significantly higher in the maternally derived cases due to a meiotic I error compared with those due to a meiotic II error. There were no significant clinical differences between patients with respect to the origin of the additional X chromosome.


A 530kb YAC contig tightly linked to the Friedreich ataxia locus contains five CpG clusters and a new highly polymorphic microsatellite.

Friedreich ataxia (FA) is a severe autosomal recessive neurodegenerative disease. The defective gene has been previously assigned to chromosome 9q13-q21 by demonstration of tight linkage to the two independent loci D9S15 and D9S5. Linkage data indicate that FRDA is at less than 1 cM from both markers. Previous physical mapping has shown that probes defining D9S15 (MCT112) and D9S5 (26P) are less than 260 kb apart and are surrounded by at least six CpG clusters within 450 kb, which might indicate the presence of "candidate" genes for FA. We isolated and characterized a 530 kb YAC (yeast artificial chromosome) contig that contains five of the CpG clusters. The YACs were used to search for new polymorphic markers needed to map FRDA precisely with respect to the cloned segment. In particular, we found a (CA)n microsatellite polymorphism, GS4, that detects 13 alleles with a PIC value of 0.83 and allows the definition of haplotypes extending over 310 kb when used in combination with polymorphic markers at D9S5 and D9S15.


Three DNA markers for hypophosphataemic rickets.

This paper presents three markers, 16D/E, pHMAI (DXS208), and CRI-L1391 (DXS274), that show close linkage for X-linked hypophosphataemic rickets (HYP). DXS274 is closely linked to HYP (theta max = 0.00, Zmax = 4.20), and DXS41 (99.6), (theta max = 0.00, Zmax = 5.20). Marker 16D/E maps distal to the disease locus (theta max = 0.05, Zmax = 3.11). The pHMAI probe recognises the same restriction fragment length polymorphism (RFLP) as 99.6. Multipoint analysis suggests that the most probable order of loci is Xpter-(DXS43, 16D/E)-HYP-DXS274-(DXS208, DXS41)-Xcen. The location of DXS274 distal to HYP cannot be excluded, as no recombinants were observed between DXS274 and HYP, or between DXS274 and DXS41/DXS208. One of the families contains a large number of recombinants, four of which are double recombinants. This most probably means that the disease in this family maps elsewhere on the X chromosome or on an autosome, indicating locus heterogeneity.


Alpha I/65 hereditary elliptocytosis in southern Italy: evidence for an African origin.

alpha I/65 Hereditary elliptocytosis (HE) is due to the duplication of TTG codon 154 (leucine) of alpha-spectrin and is associated with a constant haplotype. It was encountered exclusively in African and American Blacks, and in North Africans. We assumed that it diffused from the Benin-Togo area to Northern Africa. We now report two South Italian families with alpha I/65 HE. The phenotype fully conformed to previous descriptions. The mode of transmission was dominant; however, the manifestations were more pronounced when the common, low expression level alpha V/41 allele occurred in trans to the alpha I/65 allele, also conforming to previous records. The mutation underlying alpha I/65 HE turned out to be, again, the duplication of TTG codon 154 and the associated haplotype was the same as that encountered previously (+-+; XbaI, PvuII, MspI). Thus, the alpha I/65 allele found in Italy must have been introduced from North Africa across the Sicilian channel and would ultimately have originated from the Benin-Togo area. It would witness the same migratory stream as that followed by the Benin type haemoglobin S allele, which is also present in Southern Italy.


PCR detection of a BclI RFLP in the G6PD gene of Caucasians.

We have calculated the frequencies of two alleles of the glucose-6-phosphate dehydrogenase gene in a randomly selected group of Caucasians. Allele 1 has a frequency of 82%, whereas that of allele 2 is 18%.


Two PstI polymorphisms for the urokinase-type plasminogen activator receptor gene (PLAUR).

The cDNA probe puPAR-2 detects two PstI polymorphisms in the urokinase-type plasminogen activator receptor gene (PLAUR). This probe and the polymorphic system are described.


Analysis of the basis of resistance and susceptibility of CD4+ T cells to human immunodeficiency virus (HIV)-gp120 induced anergy.

The resistance and susceptibility of T cells to human immunodeficiency virus (HIV)-gp120 induced anergy was examined. Antigen-dependent proliferation of polyclonal T cells was markedly inhibited by gp120, whereas from the analysis of monoclonal populations, T cells resistant to the effects of gp120 could be identified. Similarly, exposure of monoclonal T cells to gp120 in the absence of accessory cells, also demonstrated that some T cells could resist the induction of anergy. Loss of antigen recognition was associated with phenotypic modulation of CD3 and CD28, which was not observed in T cells resistant to functional inactivation by gp120. Modulation of CD4 was not related to induction of anergy in the monoclonal T cells examined in this study. Inhibition of T-cell responses by anti-CD4 antibodies was compared to that by gp120. Anti-CD4 antibodies, which cross-compete with gp120 for binding to CD4, inhibited the response to antigen of monoclonal T cells. In contrast, no tolerogenic signals were delivered by pretreating T cells with the anti-CD4 antibodies in the absence of accessory cells, indicating that inhibition was due to abrogation of the interaction of CD4 with major histocompatibility complex (MHC) class II molecules expressed on accessory cells. Although the free CD4-binding region peptide of gp120 could inhibit polyclonal T-cell responses, only the carrier-bound peptide was able to modulate cloned T cells, suggesting a conformational requirement for functional inactivation through engagement of CD4. The results reported here using clonal CD4+ T-cell populations demonstrate that effects of gp120 on antigen-dependent proliferation are not uniform, and that therapeutic intervention might be directed at T-cell populations identified as susceptible to HIV-gp120 induced anergy.


CD4+ T cells control measles virus infection of the central nervous system.

CD4+ T-cell lines with specificity for individual measles virus (MV) structural proteins were obtained from immunized Lewis rats. Isolated viral proteins, either purified from virions or bacterially expressed were used as antigens for immunological assays. All the cell lines secreted interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), but were only weakly cytotoxic to autologous MV-infected astrocytes. When cultured together with memory splenic B lymphocytes these T cells did not induce secretion of MV-specific antibodies. The in vivo function of the T-cell lines was investigated in our MV-encephalitis model in the Lewis rat. Within 24 hr of intracerebral infection, adoptive transfer of single MV protein-specific T cells either decreased or prevented the subsequent clinical and histological disease depending on the MV-protein specificity of the cell lines. Furthermore, there was an earlier and enhanced viral clearance from the CNS, without a change in the anti-MV antibody titres of serum and cerebrospinal fluid (CSF) of the recipients and the control-infected animals. Prior depletion of CD8+ T lymphocytes in the recipient animals did not abrogate the protection conferred by CD4+ T-cell lines, indicating that the acute viral CNS disease is being efficiently controlled by virus-specific CD4+ T cells.


The organotin-induced thymus atrophy, characterized by depletion of CD4+ CD8+ thymocytes, is preceded by a reduction of the immature CD4- CD8+ TcR alpha beta-/low CD2high thymoblast subset.

Thymic changes in the rat induced by the thymus atrophy-inducing organotin compound di-n-butyltin dichloride (DBTC) were examined using FACS analyses. The number of CD4+CD8+ thymocytes was reduced by DBTC treatment from Day 2 onwards and reached minimum level on Days 4 and 5 after dosing. On these days the CD4-CD8- and both the CD4-CD8+ and CD4+CD8- subsets were not affected. On Day 2 we observed a reduced proportion of transferrin receptor (CD71)-positive CD4-OX44- cells, representing the cycling immature CD4-CD8+ cells, and of CD71+OX44- cells, representing the cycling CD4+CD8+ cells, but not of CD71+CD4-CD8- cells. When compared to controls, the FSChigh cell population of DBTC-treated rats contained less CD4-OX44- and OX44- cells, which were further characterized as CD2high and T-cell receptor (TcR)alpha beta- low. Moreover, fewer TcR alpha beta high cells were detected in the OX44- thymoblast subset of DBTC-treated rats. The number of CD4-CD8- thymoblasts appeared marginally decreased while the numbers of CD4+OX44+ cells, representing mature CD4+ cells, were not affected. These data indicate that DBTC causes a preferential initial depletion of immature CD4-CD8+CD2high TcR alpha beta-low thymoblasts. This initial event may result in a decreased formation of CD4+CD8+ thymoblasts and of small CD4+CD8+ thymocytes. These characteristics of the initially depleted subset indicate a specific anti-proliferative effect of DBTC and may give clues for the mechanism involved in the induction of thymus atrophy.


Role of adhesion molecules in lymphokine-activated killer cell killing of bladder cancer cells: further evidence for a third ligand for leucocyte function-associated antigen-1.

The lysis of eight human bladder cancer cell lines by lymphokine-activated killer cells (LAK) was found to be partially dependent upon the expression by the target cell of either intercellular adhesion molecule-1 (ICAM-1) or intercellular adhesion molecule-2 (ICAM-2). Using adhesion blockade studies these molecules were found to contribute towards sensitivity to lysis. Tumour lines of low grade (G1) did not constitutively express ICAM-1, but were found to express ICAM-2. High grade cells (G2, G3), however, only constitutively expressed ICAM-1 on their cell surface. Interferon-gamma (IFN-gamma) induced and augmented the expression of ICAM-1 by all except one of the cell lines (UMUC3) in a dose- and time-dependent manner. This was accompanied by an increased susceptibility to lymphokine-activated killer mediated cytolysis. Anti-ICAM-1 antibodies partially inhibited the increase in cell lysis due to IFN-gamma. However, this inhibition was not complete. When effector cells were treated with antibodies to leucocyte function-associated antigen-1 (LFA-1) the inhibition of lysis was greater and ranged from 72 to 96% with a mean of 87% inhibition. These results suggest that the increased sensitivity of IFN-gamma-treated bladder cancer cell lines to LAK cells is partially attributable to the induction of ICAM-1. However, blocking of ICAM-1 with antibodies could only partially inhibit the increased LFA-1-dependent lysis. This supports recent evidence for the existence of an additional ligand for LFA-1, other than ICAM-1 and ICAM-2.


Expression of interleukin-6 receptor on blood lymphocytes without in vitro activation.

A monoclonal antibody against the interleukin-6 receptor (IL-6R) has been used in a high-sensitivity immunofluorescence technique to study receptor expression on unstimulated blood lymphocytes. Most CD4 cells express IL-6 receptor, whilst a small and variable proportion of CD8 and B cells are positive. CD4+ cells express higher levels of receptor than CD8+ T cells, and CD45RO+ cells express higher levels than CD45RA cells.


Molecular probe analysis of Shigella dysenteriae type 1 isolates from 1940 to 1987.

Fourteen strains of Shigella dysenteriae type 1 (Shiga bacillus) isolated from people in diverse locations from 1940 to 1987 were studied. Southern hybridization with three cloned Escherichia coli genes, Shiga-like toxin I (SLTI), frd, and ompF, was used to determine restriction fragment length polymorphism (RFLP) of the genomic DNA of these strains. Digestion with each of four restriction endonucleases generated fragments of identical size to which the frd and ompF hybridized for each of the 14 strains, indicating the conservation of these genes and their flanking sequences. In contrast, after digestion with HindIII, EcoRV, and ClaI and probing with SLTI, there were RFLP among the strains. The results showed three clones of the Shiga bacillus, and suggested that dissemination of a single clone may continue for decades within a wide geographical area.


Insect peptide hormones, an overview of the present literature.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.


Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.


Hb City of Hope [beta 69(E13)Gly----Ser] in Italy: association of the gene with haplotype IX.

Hb City of Hope [beta 69(E13)Gly----Ser] was detected by reversed phase high performance liquid chromatography in an asymptomatic carrier from Naples, Southern Italy. The amino acid substitution, identified by fast atom bombardment mass spectrometry, was due to a TGG----TGA substitution as assessed by DNA sequencing. Analysis of the chromosomal background indicates that the globin gene cluster containing the mutant gene has most probably been rearranged by a recombination event, since the mutation was associated with restriction fragment length polymorphism haplotype IX, instead of haplotype I, as previously reported.


An immunohistochemical study of the catecholamine synthesizing enzymes and neuropeptides in the female guinea-pig uterus and vagina.

The uterus and vagina of the guinea pig have been examined, region by region, for acetylcholinesterase, tyrosine hydroxylase, dopamine beta-hydroxylase and aromatic amino acid decarboxylase activity, as well as for the neuropeptides, neuropeptide Y, vasoactive intestinal peptide, substance P, enkephalin and somatostatin. No acetylcholinesterase activity was localized in the uterus, though it was present in associated paracervical ganglion tissues. Of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase activity was found virtually throughout the reproductive tract, whereas aromatic amino acid decarboxylase activity was restricted in its distribution. Neuropeptide distribution was quite varied. Neuropeptide Y was found throughout the endometrium/submucosa but only in the muscularis of the vagina and not in the myometrium. Substance P was localized in the vagina and uterine horn, though not the body of the uterus. Vasoactive intestinal peptide was present in all regions of the endometrium/submucosa, but not in the myometrium of the uterine horn. Enkephalin and somatostatin were not localized in any part of the reproductive tract examined, apart from paracervical ganglion tissues. The types and significance of the nerves supplying the reproductive tract are discussed.


Hypothalamic neurotransmitter concentrations and meat quality in stressed pigs offered excess dietary tryptophan and tyrosine.

Pale, soft, exudative (PSE) pork occurs, for the most part, from environmental stress on the pig. Amino acid intake may be related to stress susceptibility through hormone and neurotransmitter induction. Two experiments were conducted to determine whether supplementation of 5 g of tryptophan (TRP) or 10 g of tyrosine (TYR) per kilogram of a 14% CP diet would alter the response of pigs to stress as measured by hypothalamic neurotransmitter concentrations and incidence of PSE. Twenty-four (Exp. 1) and 36 (Exp. 2) 92-kg pigs were offered one of three diets: control, TRP-, or TYR-supplemented for 5 d before slaughter. Dietary TRP or TYR supplementation in Exp. 1. doubled (P less than .05) plasma TRP and TYR concentrations, respectively, and increased (P less than .05) 5-hydroxytryptamine, dihydroxyphenyl ethylamine, dihydroxyphenyl acetic acid, and homovanillic acid concentrations in the hypothalamus. Pigs that exhibited stress at slaughter had lower (P less than .05) hypothalamic concentrations of epinephrine, norepinephrine, and 5-hydroxytryptamine. In Exp. 2, pigs were trucked 55 km to a commercial meat packing facility and slaughtered without a rest period. This handling procedure was designed to invoke a high incidence of PSE pork and thus be a strong test of treatments. Supplemental dietary amino acids seemed to alter the frequency distribution of the severity of PSE pork. These data indicate that dietary manipulation of amino acid precursors of neurotransmitters may offer a practical means of reducing stress response in swine.


Gamma glutamyl transpeptidase in meningitis.

Gamma glutamyl transpeptidase (GGTP) was measured serially in cerebrospinal fluid (CSF) and serum in 23 cases of meningitis (15 pyogenic and 8 tuberculous meningitis) and an equal number of age and sex matched healthy controls, to find out its diagnostic and prognostic significance in meningitis. GGTP activity was significantly elevated (p less than 0.001) in CSF and serum in meningitis as compared to control subjects. Levels were significantly higher in pyogenic as compared to tuberculous meningitis (p less than 0.001) and in CSF than in serum (p less than 0.001). The maximum elevation was seen on the 1st day and thereafter the activity declined in the majority (65.2%) of cases. However, in 3 cases of pyogenic meningitis and 5 cases of tuberculous meningitis, the GGTP activity on subsequent estimation increased serially; all these 8 cases died. It is concluded that CSF GGTP activity is significantly elevated in meningitis and serial rise in its activity is associated with poor prognosis and even fatal outcome.


Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation.

We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.


Dephosphorylation of the guanylyl cyclase-A receptor causes desensitization.

Atrial natriuretic peptide (ANP) binds to the guanylyl cyclase-A (GC-A) receptor found in tissues such as the kidney and adrenal gland, resulting in marked elevations of the intracellular signaling molecule, cGMP. Here, GC-A is shown to exist as a phosphoprotein when expressed in human embryonic 293 cells. The 32P is principally associated with phosphoserine, with only trace amounts of phosphothreonine. The addition of ANP causes a time-dependent dephosphorylation of the receptor, as well as desensitization, which is not due to an ANP-mediated decrease in the amount of receptor protein. The mobility of GC-A on sodium dodecyl sulfate-polyacrylamide gel electrophoresis increases after treatment of cells with ANP, and protein phosphatase 2A induces the same mobility shift. The protein phosphatase also catalyzes dephosphorylation of GC-A, and this is directly correlated with decreases in ANP-stimulatable guanylyl cyclase activity. Okadaic acid, an inhibitor of protein phosphatase 2A, blocks both the dephosphorylation and the desensitization. Therefore, in contrast to many other cell surface receptors, GC-A is desensitized by ligand-induced dephosphorylation.


Immunoprecipitation of alpha 2a-adrenergic receptor-GTP-binding protein complexes using GTP-binding protein selective antisera. Changes in receptor/GTP-binding protein interaction following agonist binding.

The nature of the interaction of cloned alpha 2a-adrenergic receptors from LLC-PK1-O clone cells with G proteins was investigated using an immunoprecipitation approach. Following solubilization of the alpha 2a receptors, antiserum 8730, which is directed against the C-terminal region of Gi alpha, immunoprecipitated alpha 2a receptor-Gi alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3) immunoprecipitated solubilized alpha 2a receptor-Gi alpha complexes, indicating that all three Gi alpha subtypes couple with the alpha 2a receptor. Antiserum 9072, which is directed against the C-terminal region of G(o)alpha, immunoprecipitated solubilized alpha 2a receptor-G alpha complexes indicating that these receptors are also coupled to G(o)alpha. Antiserum 8132, which is directed against G beta 36, immunoprecipitated solubilized alpha 2a receptors while the G beta 35 antiserum 8129, did not, indicating that alpha 2a receptors selectively associate with G beta 36. The binding of the partial agonist p-aminoclonidine to the solubilized alpha 2a receptor alters the association of the receptor with G proteins. Following p-aminoclonidine binding to the solubilized alpha 2a receptor, the ability of the C-terminal directed G alpha antisera 8730 and 9072 to coimmunoprecipitate the alpha 2a receptor-G alpha complex was greatly reduced. The effect of p-aminoclonidine was concentration dependent, mimicked by the full agonist UK 14304 and blocked by the alpha 2 receptor antagonist yohimbine. In contrast, antisera directed against internal regions of Gi alpha and G(o)alpha, immunoprecipitated the agonist-bound and agonist-free alpha 2a receptor equally well. These findings indicate that following the binding of agonists to the alpha 2a receptor, Gi alpha and G(o)alpha remain physically associated with the receptor but either the conformation of G alpha linked to the receptor or the conformation of the receptor itself is modified such that the epitope for the C-terminal directed anti-Gi alpha and anti-G(o)alpha antisera are not accessible. These agonist-induced conformational changes in the alpha 2a receptor-G alpha complex may be important for the activation of the G protein and the stimulation of the alpha 2a receptor signal transduction pathway.


Free amino acid dynamics in marine methanogens. beta-Amino acids as compatible solutes.

Methanogenic archaebacteria respond to osmotic stress by accumulating a series of organic molecules which function as compatible solutes. In two strains of marine methanogenic archaebacteria, Methanogenium cariaci and Methanococcus thermolithotrophicus, four key organic solutes are observed: L-alpha-glutamate, beta-glutamate, N epsilon-acetyl-beta-lysine, and betaine. The first three of these are synthesized de novo; betaine is transported into the Mg. cariaci cells from the medium. Mesophilic Mg. cariaci will preferentially transport betaine from the extracellular medium if it is present to counterbalance the external NaCl. In its absence it synthesizes N epsilon-acetyl-beta-lysine as the dominant osmolyte. This zwitterionic compound occurs at levels in Mg. cariaci which are considerably greater (based on mumol/mg of protein) than in Mc. thermolithotrophicus grown in media of the same ionic strength. Intracellular potassium ion concentrations, determined by 39K NMR spectroscopy and atomic absorption, differ significantly in the two cells. In Mc. thermolithotrophicus, intracellular K+ is balanced by the total concentration of anionic amino acid species, glutamate, and beta-glutamate. Turnover of the organic solutes has been monitored using 13C-pulse/12C-chase, and 15N-pulse/14N-chase experiments. Both beta-amino acids exhibit slower turnover rates when compared to L-alpha-glutamate or aspartate, consistent with their roles as compatible solutes. Biosynthetic information for the beta-amino acids is also provided by 13C-labeling experiments. beta-Glutamate shows a lag in 13C uptake from 13CO2, indicative of its biosynthesis from a precursor (probably a macromolecule) not in equilibrium with the soluble L-alpha-glutamate pool. Confirmation of a novel route for beta-glutamate synthesis and the production of the beta-lysine moiety from the diaminopimelate pathway is deduced from [13C2]acetate labeling patterns.


Monoclonal antibodies against the extracellular domain of the erbB-2 receptor function as partial ligand agonists.

In this paper we describe the isolation and characterization of four monoclonal antibodies (FRP5, FSP16, FWP51, and FSP77) which specifically recognize the human erbB-2 protein. All of the antibodies recognize epitopes on the extracellular domain of the receptor protein. FRP5 and FSP16 compete with one another for binding while FWP51 and FSP77 each recognize a different epitope. The effects of the antibodies on the erbB-2 receptor protein have been analyzed. Two different erbB-2-expressing cell lines, SKBR3 breast tumor cells and HC11 R111 cells, were examined. The SKBR3 cells express approximately 1 x 10(6) molecules of the erbB-2 protein/cell; HC11 R111 cells, a clone of mouse mammary epithelial cells derived by transfection of a human erbB-2 expression plasmid, contain 10-fold less erbB-2 protein than the SKBR3 cells. Treatment of the two cell lines with FRP5, FSP16, and FWP51 led to a rapid increase in the phosphotyrosine content of the erbB-2 protein. Three of the antibodies, FRP5, FSP16, and FSP77, stimulated the turnover of the erbB-2 protein. Binding of the antibodies did not stimulate DNA synthesis in HC11 R111 cells. Thus, the erbB-2-specific monoclonal antibodies behave as partial ligand agonists. The antibodies were examined for their effects upon the growth of SKBR3 and HC11 R111 cells. The growth of SKBR3 cells was inhibited by 90% following long term treatment of the cells with FSP77.


Only three mutations account for almost all defective alleles causing adenine phosphoribosyltransferase deficiency in Japanese patients.

We analyzed mutant alleles of adenine phosphoribosyltransferase (APRT) deficiency in Japanese patients. Among 141 defective APRT alleles from 72 different families, 96 (68%), 30 (21%), and 10 (7%) had an ATG to ACG missense mutation at codon 136 (APRT*J allele), TGG to TGA nonsense mutation at codon 98, and duplication of a 4-bp sequence in exon 3, respectively. The disease-causing mutations of only four (3%) of all the alleles among Japanese remain to be elucidated. Thus, a diagnosis can be made for most of the Japanese APRT-deficient patients by identifying only three disease-causing mutations. All of the different alleles with the same mutation had the same haplotype, except for APRT*J alleles, thereby suggesting that alleles with the same mutation in different families were derived from the same ancestral gene. Evidence for a crossover or gene conversion event within the APRT gene was observed in an APRT*J mutant allele. Distribution of mutant alleles encoding APRT deficiency among the Japanese was similar to that seen in cystic fibrosis genes among Caucasians and Tay-Sachs genes among the Ashkenazi Jews.


Recombinant interleukin-12 suppresses the synthesis of immunoglobulin E by interleukin-4 stimulated human lymphocytes.

Interleukin-12 is a recently discovered lymphokine displaying an array of in vitro activities suggesting a major role in protective immunity against infectious agents like viruses. This study provides evidence that IL-12 may also be implicated in the selection of the immunoglobulin isotypes. We show that picomolar concentrations of rIL-12 markedly inhibit the synthesis of IgE by IL-4-stimulated PBMC. The suppression of IgE is observed at the protein and at the mRNA levels, it is isotype specific, and it is abolished by neutralizing anti-IL-12 mAbs. IL-12 may suppress IgE synthesis by: (a) inducing the production of IFN-gamma, a known inhibitor of IgE synthesis and (b) by a novel mechanism which is IFN-gamma independent. The best evidence for this is from studies on IgE synthesis by IL-4-plus hydrocortisone-stimulated umbilical cord blood lymphocytes, which do not produce detectable amounts of IFN-gamma. In such cultures, rIL-12 inhibits IgE synthesis even in the presence of a large excess of neutralizing anti-IFN-gamma mAb.


Papillon-Lefèvre syndrome. Characterization of peripheral blood and gingival lymphocytes with monoclonal antibodies.

A 14-year-old boy with typical features of Papillon-Lefevre syndrome (PLS) is presented. The purpose of this report was to study the immunopheno-typic features of the peripheral blood and gingival tissue lymphocytes with monoclonal antibodies in the patient. Peripheral blood T-cells, helper-T cells, suppressor-T cells, HLA-DR+ cells and IL-2R+ cells were determined using appropriate monoclonal antibodies and indirect immunofluorescence methods. B-cells were identified using the direct immunofluorescence technique. The gingival tissue was processed for both histopathological and immunohistological examinations. Gingival tissue lymphocytes were identified using monoclonal and polyclonal antibodies with the immunoperoxidase technique. Although we have not detected any significant alterations in the peripheral blood B-cell and T-cell populations, NK cells were significantly increased. HLA-DR+ cells and IL-2R+ cells were within normal limits. Histopathology of the diseased tissue revealed predominance of plasma cells in the lamina propria. The majority of the plasma cells were bearing IgG isotype. Most of the CD3+ T-cells were located beneath the pocket epithelium with an almost equal distribution of CD4+ and CD8+ T-lymphocytes, in situ. These findings indicate that PLS is a IgG+ plasma cell dominated lesion with the participation of T-lymphocytes, having similar distributions of both subsets. While the etiopathogenesis of the syndrome still has to be elucidated, these immunohistological findings could be used for further studies in this intriguing entity.


Terazosin: a new alpha adrenoceptor blocking drug.

Terazosin (Hytrin; Abbott Laboratories, North Chicago, IL) is a new, selective alpha 1-adrenoceptor blocking agent used on once-a-day basis for therapy of mild-to-moderate hypertension. Its pharmacologic properties are similar to those of prazosin. Terazosin however, differs from prazosin in that its water solubility is 25 times greater than that of prazosin and its elimination half-life is about three times that of prazosin. Greater water solubility facilitates intravenous formulation, and longer half-life allows once-daily administration of terazosin. Terazosin is effective in lowering blood pressure and has a beneficial effect on plasma lipid profile. The major advantage of terazosin compared with prazosin, however, is its long duration of action. Terazosin is safe and effective when used in combination with diuretics and other antihypertensive agents, and in the long-term treatment of patients with mild to moderate essential hypertension.


Physical restraint use and cognitive decline among nursing home residents.

This study investigated the association between physical restraint use and decline in cognition. Cohort analytic study describing changes in resident characteristics. Eight nursing homes, both urban and suburban, operated by a proprietary corporation in a large metropolitan area. 437 nursing home admissions, with 201 remaining at 1 year. Cognitive status was measured by geropsychiatrists, using the Folstein Mini-Mental State Exam, during a psychiatric evaluation of the resident. Daily restraint use was documented from nursing orders. Observations were made at 2 weeks, 10 weeks, and 1 year. Restraint use alone and in combination with neuroleptic use was associated with poor cognition. Other variables associated with poor cognitive scores were: ADL impairment, poor adaptive behavior, and longer time in the nursing home. The use of neuroleptics alone was not significant. Variables which were associated with good cognitive status were: being non-ambulatory but without dementia and having strong social support. These findings raise the possibility that restraint use may contribute to cognitive impairment, specifically among residents who have moderate to no cognitive impairment at admission; however, the findings do not exclude an alternative explanation that residents undergoing cognitive decline are more likely to be put in restraints. Further research is needed to understand whether factors which can be manipulated contribute to cognitive decline.


Microbial contamination of brushes used for preoperative shaving.

Microbial contamination of brushes used for preoperative shaving was investigated. Of the 24 brushed examined, 18 were contaminated with 10(6)-10(9) colony forming units (cfu) per brush. Non-fermentative Gram-negative bacilli such as Pseudomonas aeruginosa and Xanthomonas maltophilia, and yeast-like fungi such as Candida parapsilosis were the primary contaminants. The mean bacterial count on the skin after the use of contaminated brushes (having a mean bacterial count 2.2 x 10(8) cfu) in 14 subjects was 4.6 x 10(5) cfu 25 cm-2, which was about 100 times (p less than 0.001) the control level. Contaminated brushes could not be disinfected with 80% ethyl alcohol, 0.1% sodium hypochlorite or 0.5% chlorhexidine. These findings suggest that the use of brushes should be avoided for preoperative shaving with a razor, and that sterile gauze and shaving foam should be used instead of a brush and soap.


Bacterial contamination of enteral feeds as a possible risk of nosocomial infection.

The degree of microbiological contamination in enteral diets was studied and the possible infectious complications that could arise in the patient after administration of an enteral feed were evaluated. Of the 208 diets studied, 56 (26.9%) were contaminated and 152 (73.1%) were sterile. Of the 56 contaminated diets, 11 could be used as delivered, but the other 45 required further modification. Of the patients who had received enteral feeding, 43 developed gastrointestinal symptoms in the first 24 h (fever, vomiting, abdominal pain and diarrhoea). Twenty-nine (67.4%) had received a contaminated diet and 14 (32.6%) an uncontaminated one. We conclude that contamination of enteral feeds may constitute a risk factor for nosocomial infection, and consider it necessary to carry out epidemiological surveillance in order to control the factors which may lead to contamination of enteral diets.


Partial characterization of an endemic strain of a methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) homogeneously resistant to beta-lactam antibiotics.

Selected strains of methicillin- and aminoglycoside-resistant Staphylococcus aureus (MARSA) were subjected to a preliminary examination. They were representative of a larger group collected in a routine clinical microbiology laboratory over a period of 2 years. MARSA was endemic in the associated hospital. The characteristics investigated were antimicrobial resistance, the production of beta-lactamase, free and bound coagulase, protein A, DNA-ase, urease, lipase and pigment. The MARSA strains were generally indistinguishable, other than in their antimicrobial resistances. The resistance to methicillin was completely homogeneous. Except with imipenem, growth extended to the edge of discs containing methicillin and the other beta-lactam antibiotics tested when the strains were cultured at 37 degrees C on media without added salt. Homogeneous resistance may confer an epidemiological advantage on strains of this phenotype.


Staphylococcus aureus still colonizes the untreated neonatal umbilicus.

Two different neonatal umbilical cord treatment regimens were studied prospectively. Although a greater proportion of cords had separated by the seventh day in those babies not treated with topical antiseptics (47% vs. 26%), there was a significant excess (53% vs. 30%) of umbilical colonization by Staphylococcus aureus compared to those neonates whose cords were treated with alcohol wipes and hexachlorophane powder. The main purpose of treating cords is to prevent significant S. aureus colonization, and therefore current proposals to stop antiseptic treatment of umbilical cords should be disregarded.


Handwashing in a neonatal intensive care nursery: product acceptability and effectiveness of chlorhexidine gluconate 4% and triclosan 1%.

The effectiveness of triclosan 1% w/v against methicillin-resistant Staphylococcus aureus (MRSA) and its effect on skin were compared with chlorhexidine gluconate 4% w/v ('Hibiclens') in a 7-week trial. Clinical information of MRSA rates obtained during the previous 10 months and results from earlier user acceptability trials were included. The average number of new cases of MRSA per week was reduced from 3.4 to 0.14 (P less than 0.0001) in the experimental ward whilst no significant changes occurred in the control ward. Staff reported less skin damage and a higher rate of acceptance with the experimental product. Based on results of the trial, a proposal to introduce triclosan for a 12-month study period has been accepted.


Evaluation of a commercial chemiluminescent gene probe system 'AccuProbe' for the rapid differentiation of mycobacteria, including 'MAIC X', isolated from blood and other sites, from patients with AIDS.

Optimal therapy of mycobacterial infections in acquired immunodeficiency syndrome (AIDS) is difficult to achieve because of the time needed for a conventional culture to differentiate between Mycobacterium avium-intracellulare complex (MAIC) and Mycobacterium tuberculosis complex (MTBC). The recent commercial availability of gene probing techniques has introduced the potential for more rapid differentiation. We have evaluated the suitability of this technique. The specificity of the chemiluminescent gene probe system 'AccuProbe' was determined for differentiating mycobacterial isolates from 114 AIDS patients. 'AccuProbe' was 100% specific for MTBC isolates (21 of 21 isolates). Using two separate probes to MAIC and 'M. avium-intracellulare complex subtype X' (MAIC-X), 'AccuProbe' was 96% specific (87 of 91 isolates) with 5% of isolates belonging to the MAIC-X group. There were no cross-reactions between any of the probes. Using a modification of the manufacturer's protocol, 'AccuProbe' was used in a 6-month trial for the rapid differentiation of mycobacteria grown from 'Bactec' blood culture. Fifty-seven isolates (31 patients) from 805 Bactec 13A blood cultures (510 patients) were investigated. Ninety-nine per cent of isolates were able to be identified after a mean incubation period of 2.1 weeks (SD 1.2 weeks). Ninety-three per cent of isolates were reported with a presumptive identification the same day and 99% by the day after the culture flagged positive.


Recovery of coliforms from the hands of nurses and patients: activities leading to contamination.

Coliform type organisms were recovered from the hands of nurses and patients in an Orthopaedic Hospital. Coliforms were frequently recovered from nurses' hands after touching patients' washing materials and clothing as well as after bed making, sluice room activities and handling clean or dirty linen and curtains. The recovery rates were higher in wards for spinally injured patients than in the surgical wards. Coliforms were recovered with similar frequencies, to those from nurses, from the hands of patients in both types of wards.


Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections.

Restriction fragment length polymorphism analysis (RFLP) of total DNA and rDNA regions was used for the epidemiological evaluation of 10 Enterobacter cloacae nosocomial isolates obtained from nine patients in our hospital. Five of these patients were hospitalized during overlapping periods, thus raising the question of cross-contamination. A single biochemical pattern and antibiotic susceptibility profile was observed for all isolates but one. In contrast, based on the results of total DNA and rDNA RFLP patterns, the genetic unrelatedness of the isolates was clearly shown, thus excluding a common source of contamination or patient-to-patient transfer.


Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.