Releasable GABA in tubular epithelium of rat kidney

Summary The distribution of gamma-aminobutyric acid (GABA) in the rat kidney was examined by immunocytochemical techniques. GABA-like immunoreactivity (GABA-LI) was predominantly confined to the renal tubules, including the ascending parts of the distal tubules, and the loops of Henle, the collecting tubules and ducts, and the connective parts of the convoluted tubules. In GABA-positive cortical tubules, about half of the epithelial cells were labelled. The labelled cell type showed the ultrastructural features of principal cells. Depolarizing stimulation by ouabain and high K⁺ concentration evoked the efflux of endogenous GABA from kidney slices. The present findings, along with previous results, suggest that GABA released from renal tubular epithelium, and transported with the urine, might be involved in the modulation of contractility in the urinary tract.     

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate

Summary Rat brain homogenate was preloaded with [³H]noradrenaline or [³H]GABA and stimulated with high K⁺. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [³H]noradrenaline release in the same range of toxin concentrations starting below 10^–10M. In contrast, release of -amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.     

Biological effects of trilostane in vitro on oocyte maturation and fertilization in the hamster

Summary The effects of the inhibition of steroidogenesis by trilostane on oocyte maturation were examined by studying spontaneous maturation and fertilization in vitro. 10^–6 M trilostane had no influence on the meiotic process, whether the oocytes were naked or not. At a concentration of 10^–6 M and 10^–7 M trilostane, low normal pronuclear formation and high polyspermy were found during in vitro fertilization. However, no retarded male pronuclear development could be detected in the trilostane-treated group. Thus, steroid producing activity within ova is apparently necessary to prevent multiple sperm penetration, but it has no effect on meiosis or the action of the so-called male pronucleus growth factor (MPGF).     

Role of glutamic acid decarboxylase in the pathogenesis of type 1 diabetes

Glutamic acid decarboxylase (GAD) is considered to be one of the strongest candidate autoantigens involved in triggering β-cell-specific autoimmunity. The majority of recent onset type 1 diabetes patients and prediabetic subjects have anti-GAD antibodies in their sera, as do nonobese diabetic (NOD) mice, one of the best animal models for human type 1 diabetes. Immunization of young NOD mice with GAD results in the prevention or delay of the disease as a result of tolerizing autoreactive T cells. Autoimmune diabetes can also be prevented by the suppression of GAD expression in antisense GAD trans genic mice backcrossed with NOD mice for seven generations. These results support the hypothesis that GAD plays an important role in the development of T-cell-mediated autoimmune diabetes. However, there is some controversy regarding the role of GAD in the pathogenesis of diabetes. Whether GAD truly plays a key role in the initiation of this disease remains to be determined. The examination of the development of insulitis and diabetes in β-cell-specific GAD knockout NOD mice will answer this remaining question. Received 12 April 2002; received after revision 24 May 2002; accepted 27 May 2002 RID="*" ID="*"Corresponding author.     

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

The involvement of the renin-angiotensin system in the regulation of cell proliferation in the rat endometrium

Oestrogens are known to enhance angiotensin biosynthesis by increasing the elaboration of its precursor, angiotensinogen. On the other hand, we found that inhibition of angiotensin-converting enzyme (ACE) suppressed the proliferative response of the rat anterior pituitary gland to oestrogens. To answer the question whether the angiotensin system is involved in the control of the cell proliferation of the uterine epithelium, the effects of an ACE inhibitor, enalapril maleate, and of angiotensins II and IV, alone or together with losartan, an antagonist of angiotensin receptor type 1 (AT1), on endometrial epithelial cell proliferation have been studied. The experiments were performed on ovariectomized female Wistar rats. In the first experiment the animals were injected with a single dose of oestradiol benzoate or received an injection of solvent only. Half of the oestrogen-treated rats were injected additionally with enalapril maleate (EN, twice daily). The incorporation of bromodeoxyuridine (BrDU) into endometrial cell nuclei was used as an index of cell proliferation. It was found that oestradiol alone dramatically increased the BrDU labelling index (LI) of endometrial cell nuclei, and this effect was partially blocked by the simultaneous treatment with EN. In the second experiment, the animals were injected intraperitoneally with angiotensin II (AII), angiotensin IV (AIV) or saline, alone or together with losartan. It was found that AIV induced an increase in the LI in uterine epithelium, and this effect was not blocked by the simultaneous treatment with losartan. The increase in LI in uterine epithelium was also observed in the rats treated with AII and with losartan. These findings suggest an involvement of angiotensin IV in the control of uterine epithelium cell proliferation. Received 12 October 1998; received after revision 6 January 1999; accepted 2 February 1999     

The functions of mucosal T cells in containing the indigenous commensal flora of the intestine

There is an immense load of non-pathogenic commensal bacteria in the distal small intestine and the colon of mammals. The physical barrier that prevents penetration (translocation) of these organisms into the body is a simple epithelium comprised of the single enterocyte/colonocyte cell layer with its overlying mucus. In this review, we discuss the roles of intestinal T cells in initiating and regulating innate and adaptive mucosal immune responses of the mucosal immune system that avoid or limit penetration of the commensal intestinal bacteria. Received 9 August 2002; accepted 9 September 2002 RID="*" ID="*"Corresponding author.     

Distribution of HIV-1 in the genomes of AIDS patients

The localization of HIV-1 proviruses in compositional DNA fractions from 27 AIDS patients during the chronic phase of the disease with depletion of CD4+ and different levels of viremia showed the following. (1) At low viremia, proviruses are predominantly localized in the GC-richest isochores, which are characterized by an open chromatin structure; this result mimics findings on HIV-1 integration in early infected cells in culture. (2) At higher viremia, an increased distribution of proviruses in GC-poor isochores (which match the GC poorness of HIV-1) was found; this suggests a selection of cells in which the isopycnic localization leads to a higher expression of proviruses and, in turn, to higher viremia. (3) At the highest viremia, integrations in GC-rich isochores are often predominant again, but generally not at the same level as in (1); this may be the consequence of new integrations from the extremely abundant RNA copies.Received 21 November 2003; received after revision 13 January 2004: accepted 15 January 2004     

Basal autophagy is involved in the degradation of the ERAD component EDEM1

Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 January 2009; received after revision 16 February 2009; accepted 17 February 2009 V. Le Fourn, K. Gaplovska-Kysela: These authors equally contributed to this work.     

Neuronal plasticity and regeneration in the olfactory system of mammals: morphological and functional recovery following olfactory bulb deafferentation

The mammalian olfactory system has the unique property in the permanent turnover of the olfactory sensory neurons under normal conditions and following injury. This implies that the topographical map of the epithelium-to-bulb connections generated during ontogenesis has to be maintained despite neuron renewal in order to insure olfactory information processing. One way to investigate this issue has been to disrupt the peripheral connections and analyze how neural connections may be reestablished as well as how animals may perform in olfactory-mediated tasks. This review surveys the main data pertaining to both morphological and functional recoveries taking place in the peripheral olfactory system following olfactory bulb deafferentation. Conclusions from these studies are enlightened by recent data from molecular biology.     

Linear regression forecasting in the presence of ar(1) disturbances

This paper is concerned with time-series forecasting based on the linear regression model in the presence of AR(1) disturbances. The standard approach is to estimate the AR(1) parameter, ρ, and then construct forecasts assuming the estimated value is the true value. We introduce a new approach which can be viewed as a weighted average of predictions assuming different values of ρ. The weights are proportional to the marginal likelihood of ρ. A Monte Carlo experiment was conducted to compare the new method with five more conventional predictors. Its results suggest that the new approach has a distinct edge over existing procedures.     

Expression of nerve growth factor-like polypeptides and immunoreactivity related to the two types of neurotrophin receptors in earthworm tissues

Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress. Received 12 November 2001; received after revision 8 January 2002; accepted 8 January 2002     

Biosynthesis of 1-alkenes in the defensive secretions ofTribolium confusum (Tenebrionidae); stereochemical implications

Summary The terminally unsaturated hydrocarbons of the defensive secretion ofTribolium confusum are biosynthesized from fatty acids by oxidative decarboxylation. The process involves an enantiospecific cleavage of the C–H bond of thepro-(S) C(3)–H atom and simultaneous decarboxylation of the acid into an 1-alkene and carbon dioxide via ananti-periplanar transition state geometry (anti-elimination). The stereochemistry of this biotranformation is identical in all respects with the same reaction in higher plants. The mechanism seems to be of general importance for the biosynthesis of many vinylic substructures of natural products from oxygen-containing precursors.     

Protective effect of Shigyaku-to,a traditional Chinese herbal medicine,on the infection of herpes simplex virus type 1 (HSV-1) in mice

The antiviral activity of Shigyaku-to (TJS-109), a traditional Chinese herbal medicine, was investigated in mice infected with herpes simplex virus type 1 (HSV-1). TJS-109 is a combination of the medicinal plant extracts fromZingiberis siccatum rhizoma,Aconiti tuber andGlycyrrhizae radix in a specific proportion. Mice infected with a 10 LD₅₀ dose of HSV-1 were treated with TJS-109 orally at doses of 1.25 to 20 mg/kg 2 days before, and 1 and 4 days after the infection. The treated groups had 80% (1.25 mg/kg), 40% (5 mg/kg) and 23% (20 mg/kg) mortality rates 25 days after the infection as compared with a 100% mortality rate in control mice treated with saline. When HSV-1 infected mice (recipients) received CD8⁺T cell fractions derived from spleens of mice treated with TJS-109 (donors), 70% of recipients survived, as compared with 0% survivors in the groups of mice treated with saline, B cell fractions, CD4⁺ T cell fractions or macrophage-enriched fractions prepared from the same donors. TJS-109 did not show any virucidal activities against HSV-1 or any virostatic activities on the growth of HSV-1 in Vero cells. These results suggest that TJS-109 protected mice exposed to lethal amounts of HSV-1 through the activation of CD8⁺ T cells.     

Hypothalamo-hypophyseal-gonadal function in the rat following administration of the novel and selective D-1 agonist CY 208-243

Summary The effects of the novel and selective dopamine D-1 agonist CY 208-243 on the rat hypothalamo-hypophyseal-gonadal (HHG) axis were studied. CY 208-243 did not modify the concentration of luteinizing hormone (LH) in serum from female or male rats, and had no effect upon opiate antagonist-induced stimulation of LH secretion in male rats. CY 208-243 did not inhibit ovulation in cycling female rats. Thus, D-1 receptor activation by systemic drug administration does not alter HHG function in rats.