High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

Avian thyroid development in chemically contaminated environments: is there evidence of alterations in thyroid function and development?

Poor reproductive success, developmental abnormalities, and behavioral alterations in fish-eating birds in some Great Lakes areas have led to more than 35 years of toxicological studies and residue monitoring of herring gull (Larus argentatus) populations. Polyhalogenated aromatic hydrocarbons (PHAHs), especially polychlorinated biphenyls (PCBs), are widespread contaminants in the Great Lakes ecosystem. The introduction of regulations and elimination of point sources since the 1970s have resulted in decreased PHAHs in fish-eating bird eggs and tissues. PCB exposure is associated with thyroid disruption (hypothyroidism) in mammals, but much less is known of PCB effects on avian thyroid function. Our 1998-2000 studies of herring gulls from the Great Lakes show that both pipping embryos and prefledglings from highly contaminated sites have marked depletion of thyroid gland hormone stores compared with similarly aged gulls at the reference sites. However, organismal hypothyroidism was not apparent in many embryo and chick collections where severe depletion of thyroid gland hormone was observed. Adults, sampled at two high PCB sites and a low PCB site in the Great Lakes and the maritime reference colony in 2001, showed no differences in organismal thyroid status across sites, but gulls from the high sites had enlarged thyroid glands and depressed thyroid gland hormone stores. Here we discuss the evidence that ecological exposure to PHAHs are responsible for thyroid deficiencies in gulls and that during development these deficiencies lead to developmental abnormalities in young gulls from highly contaminated Great Lakes sites.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Cationic dependency of high affinity prostaglandin F2α receptors in bovine corpus luteum cell membranes

The specific binding of [³H] Prostaglandin (PG) F₂α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca²⁺ (HCa²⁺) was examined. The membranes prepared in H-EDTA buffer bound less [³H] PGF₂α and had a single class of PGF₂α receptors with an apparent dissociation constant (Kd) of 2.7 × 10^?8M. The addition of Ca²⁺ to these membranes resulted in increased binding with the appearance of new PGF₂α receptors of Kd = 4.3 × 10^?9M. The membranes prepared in HCa²⁺ buffer contained two classes of receptors with Kds = 2.9 × 10^?9M and 2.9 × 10^?8M. The removal of Ca²⁺ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10^?9M. These results suggest the dependency of high affinity PGF₂α receptors, in bovine corpus luteum cell membranes, on cations.     

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

As in the tobacco hornworm Manduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone.When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.     

L-methionine γ-lyase from Citrobacter freundii: Cloning of the gene and kinetic parameters of the enzyme

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.     

An extended developmental study of γ-glutamyltranspeptidase in rat liver plasma membranes: identification of specific patterns of changes in activity in the adult as well as the neonatal state

Summary Homogenates and plasma membranes were isolated from the livers of male Fischer 344 rats ranging in age from 19 hr to 92 days postnatal. These plasma membranes exhibited comparable levels of purity: protein yields were 2–2.5%; relative specific activities of 5-nucleotidase and ouabain-sensitive Na⁺/K⁺-ATPase were from 8–11 and from 12–19, respectively. 5-nucleotidase and ouabain-sensitive Na⁺-K⁺-ATPase displayed distinct and different developmental patterns. The activity of -glutamyltranspeptidase was found to be at exceptionally high levels in isolated plasma membranes immediately after birth and to decline precipitously thereafter achieving and maintaining low levels from days 3–21 postnatal. Liver plasma membrane -glutamyltranspeptidase activity was observed to increase 9.2 fold from this low point, first rising on day 21, peaking on day 40 and returning to low levels by day 56. From day 56 day to 92 postnatal, -glutamyltranspeptidase activity was expressed at a uniformly low level but a level 2 fold higher than that preceeding the rise at day 40. The hormone determinants of these developmental changes in -glutamyltranspeptidase activity are discussed.     

Oxygen levels and the regulation of cell adhesion in the nervous system: A control point for morphogenesis in development,disease and evolution?

In this article, I discuss the hallmarks of hypoxia in vitro and in vivo and review work showing that many types of stem cell proliferate more robustly in lowered oxygen. I then discuss recent studies showing that alterations in the levels and the types of cell and substrate adhesion molecules are a notable response to reduced O₂ levels in both cultured primary neural stem cells and brain tissues in response to hypoxia in vivo. The ability of O₂ levels to regulate adhesion molecule expression is linked to the Wnt signaling pathway, which can control and be controlled by adhesion events. The ability of O₂ levels to influence cell adhesion also has far-reaching implications for development, ischemic trauma and neural regeneration, as well as for cancer and other diseases. Finally I discuss the possibility that the fluctuations in O₂ levels known to have occurred over evolutionary time could, by influencing adhesion systems, have contributed to early symbiotic events in unicellular organisms and to the emergence of multicellularity. It is not my intention to be exhaustive in these domains, which are far from my own field of study. Rather this article is meant to provoke and stimulate thinking about molecular evolution involving O₂ sensing and signaling during eras of geologic and atmospheric change that might inform modern studies on development and disease.     

Immunocytochemical localization of POMC-derived peptides (adrenocorticotropic hormone, α-melanocyte-stimulating hormone and β-endorphin) in the pituitary,brain and olfactory epithelium of the frog,Rana esculenta,during development

Developmental stages of Rana esculenta, starting with the posterior limb-bud stage (stage 26) up to a few days after metamorphosis, were examined immunohistochemically to localize cells and fibers producing some POMC-derived peptides, namely, -MSH, ACTH and -END. Anti ACTH and anti -MSH revealed a positive reaction in the pars intermedia during all stages of development included in this study, whereas no immunoreactivity in this pituitary zone was ever evidenced with anti -END. In the pars distalis strongly positive cells were seen with anti ACTH and anti -END, while anti -MSH yielded weakly positive cells. Interestingly, these peptides were colocalized in the same cells. Immunoreactivity for -MSH was no longer present in the pars distalis during metamorphic climax and postmetamorphosis. In the brain of premetamorphic tadpoles, belonging to stages 26 to 30, a few neurons in the posterior telencephalon showed a positive reaction only with anti -MSH,but from stage 31 (prometamorphosis) onwards, ACTH and -endorphin-like peptide producing cells, together with -MSH-immunoreactive cells, were seen in this region and in the anterior preoptic area and infundibulum. This situation persisted in the subsequent stages of development. Anti -MSH also revealed weakly positive cells in the olfactory epithelium in premetamorphic tadpoles; strong immunoreactivity with anti -MSH was seen in olfactory epithelium cells in animals during prometamorphosis, metamorphic climax and postmetamorphosis. The possible significance of these findings is briefly discussed.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

Site of action for β-endorphin-induced changes in plasma luteinizing hormone and prolactin in the ovariectomized rat

A number of sites have been hypothesized as loci at which opioid substances act to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) (1–8). The aim of the present study was to determine the site(s) at which the opioid peptide β-endorphin (β-END) acts to influence plasma LH and PRL levels in the ovariectomized (OVX) rat. β-END, administered into the third ventricle of conscious OVX rats fitted with jugular catheters, significantly decreased plasma LH in doses ? 50 ng and increased PRL levels at all doses administered (10, 50, 100 and 250 ng) in a dose dependent fashion. To identify possible central nervous system sites of action, 250 ng β-END was unilaterally infused into various brain sites. Plasma LH was significantly decreased and plasma PRL significantly increased by infusions into the ventromedial hypothalamic area, the anterior hypothalamic area, and the preoptic-septal area. There was no significant effect of β-END infusions into the lateral hypothalamic area, amygdala, midbrain central gray, or caudate nucleus. When hemipituitaries of OVX rats were incubated $\text{in}$$\text{vitro}$ with β-END (10^?7M to 10^?5M), there was no suppression of basal or LHRH-induced LH release, nor was there any alteration of basal PRL release. It is concluded that β-END acts at a medial hypothalamic and/or preoptic-septal site and not the pituitary, to alter secretion of LH and PRL.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.     

α-Adrenergically mediated changes in membrane lipid fluidity and Ca2+ binding in isolated rat liver plasma membranes

Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca²⁺ concentration by mobilizing Ca²⁺ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na⁺-oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg²⁺, reducing free Ca²⁺ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca²⁺ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of ⁴⁵Ca²⁺ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca²⁺-mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca²⁺ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Mammalian DSCAMs: roles in the development of the spinal cord,cortex, and cerebellum?

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral-dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal-Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal-ventral patterning and cell-fate specification in the developing CNS.