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The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterize ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 minutes of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 minutes following withdrawal from chronic ethanol exposure (0.50% v/v). We characterize the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on 3 domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish.

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.

1. Normal differentiation of PC 12 cells and dorsal root ganglionic neurons in culture need nerve growth factor (NGF) for their neurite outgrowth. 2. An antibody against GM3 ganglioside was found to inhibit the nerve growth factor mediated neurite formation of both the cells in vitro significantly. 3. Further analysis revealed that the binding of 125I-NGF to live PC 12 cells could be markedly inhibited by anti-GM3 antibody in a dose dependent manner. 4. Scatchard analysis revealed that in the presence of anti-GM3 antibody only some low affinity binding sites were available for NGF-high affinity binding sites were totally blocked. 5. These results further strengthen the hypothesis that anti-GM3 antibody affects neuronal cell growth by interfering with the coupling of growth factors to their cell surface receptors.

A cross sectional study of 116 Santal girls was undertaken to compare the relationship of biceps and triceps skinfolds, waist, hip and mid upper arm circumferences, waist-hip ratio and conicity index with three body composition variables; percentage of body fat, fat mass and fat free mass of the anthropometric characteristics. Waist circumference displayed the strongest correlations (p,0.001) with present body fat (r=0.85) and fat mass (r=0.86). Waist-hip ratio did not significant correlation with any measure of body composition. The correlation of conicity index with body composition measures was intermediate between those of waist circumference and waist-hip ratio. Results also revealed that the greatest amount of variation of percentage of body fat (73.1%) and fat mass (75.5%) was explained by waist circumference. In conclusion the present study showed that waist circumference is much more strongly associated with body fat than other anthropometric measures including waist-hip ratio and conicity index. Thus the use of waist circumference instead of waist-hip ratio or conicity index could be advantageous in epidemiological and clinical studies which deals with the relationship of central adiposity with percentage of body fat and fat mass.

The zebrafish is increasingly utilized in behavioral brain research, as it offers a useful compromise between system complexity and practical simplicity. However, a potential drawback of this species in behavioral research is that individuals are difficult to distinguish. Here we describe a simple marking procedure, subcutaneous injection of color dyes, that may alleviate this problem. The procedure allowed us to successfully mark zebrafish and distinguish them for a period of more than 30 days, which is sufficiently long for most behavioral paradigms developed for this species. In addition, we also provide data suggesting that the injection-based marking does not significantly alter social interaction, as defined by the frequency of agonistic behaviors within shoals.

Fetal Alcohol Spectrum Disorder (FASD) is a preventable disease of the child resulting from alcohol (ethanol) consumption by pregnant women. Despite being preventable, FASD represents a prevalent problem throughout the world. Embryonic alcohol induced abnormalities in behavioral responses to social stimuli have been shown in humans and zebrafish. The neurobiological mechanisms underlying the abnormalities remain obscured. Here we start a mechanistic analysis by investigating the effect of embryonic alcohol exposure on the neurochemistry of zebrafish. The differeing severity of symptoms seen in FASD may be partially due to genetic factors. To explore such genetic effects, here we analyzed two distinct zebrafish strains: AB and TU. Zebrafish were exposed to one of the following concentrations of alcohol, 0.00%, 0.25%, 0.50%, 0.75%, 1.00% (vol/vol %) at 24 hours post-fertilization (hpf) for 2 hours. From whole brain extracts we analyzed the amount of neurotransmitters dopamine and serotonin and their metabolites across 4 different developmental time points: 15, 40, 70 and 102 days post- fertilization (dpf) using high performance liquid chromatography (HPLC). AB zebrafish exhibited a significant dose dependent embryonic alcohol exposure effect which increased in robustness with age. However, TU showed no such concentration effect: the levels of neurochemicals remained mainly unaltered by embryonic alcohol exposure in all age groups. We also analyzed the amount of alcohol reaching the embryo in the two strains and ruled out the possibility that TU has a more protective chorion. We conclude that the uncovered strain differences are due to genetic differences that protect TU from the deleterious effects of embryonic alcohol exposure.

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist-induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist-induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species.

The zebrafish is becoming increasingly popular in behavior genetics because it may allow one to conduct large scale mutation and drug screens facilitating the discovery of mechanisms of complex traits. Strain differences in adult zebrafish behavior have already been reported, which may have important implications in neurobehavioral genetics. For example, we have found the AB and SF strains to differ in their behavioral responses to both acute and chronic alcohol exposure. In the current study, we further characterize these strains using semi-quantitative RT-PCR to measure the expression of ten selected genes and HPLC to measure the levels of nine neurochemicals. We chose the target genes and neurochemicals based upon their potential involvement in alcohol and other drugs of abuse related mechanisms. We quantified the expression of the genes encoding D1-R, D2a-R, D4a-R dopamine receptors, GABA(A)-R, GABA(B)-R1, GAD1, MAO, NMDA-R (NR2D subunit), 5HT-R1bd and SLC6 a4a. We found the gene encoding D1 dopamine receptor over-expressed and the genes encoding GABA(B1) receptor and solute family carrier protein 6 (SLC6) 4a under-expressed in SF compared to AB. We also found the level of all (dopamine, DOPAC, Serotonin, GABA, Glutamate, Glycine, Aspartate, Taurine) but one (5HIAA) neurochemicals tested decreased in SF as compared to AB. These results, combined with previously identified behavioral differences between the AB and SF strains, demonstrate the importance of strain characterization in zebrafish. They now also allow formulation of working hypotheses about possible mechanisms underlying the differential effects of acute and chronic alcohol treatment on these two zebrafish strains.

Although androgen receptor (AR) within myocytes is thought to mediate many of the effects of testosterone and other androgens on skeletal muscle, little is known about the functions of AR within these cells. We, therefore, studied the ultrastructure of skeletal muscle of HSA-AR transgenic (Tg) mice that overexpress AR selectively in myocytes and exhibit neuromuscular atrophy. We examined male HSA-AR mice from two different founding lines: L78 (lower copy number and less severe phenotype) and L141 (higher copy number and more severe phenotype) and compared these to wild-type (Wt) brothers. We also examined testosterone-treated female mice from these two lines and compared them both to their Wt sisters and to vehicle-treated controls. Ultrastructural examination of extensor digitorum longus sections using transmission electron microscopy revealed remarkably disorganized myofibrils in male Tg and testosterone-treated female Tg mice. Quantification of ultrastructural pathology indicated reduced myofibril width, hypertrophic and hyperplastic intermyofibrillar mitochondria, and pronounced glycogen accumulation in HSA-AR males of both lines. Reduced myofibrillar width and increases in mitochondrial number, size, and volume density were also observed in testosterone-treated HSA-AR females, although glycogen accumulation was not observed. Structural abnormalities in mitochondria were also associated with increases in electron transport chain activity and systemic resting metabolic rate, indicative of hypermetabolism. We find that overexpression of AR in myocytes of HSA-AR mice results in alterations in myofibrils, mitochondria, and glycogen. Alterations in myofibrils and mitochondria appear to result from acute actions of testosterone, whereas those on glycogen do not. Pathology of myofibrils and/or mitochondria may, therefore, mediate in part the neuromuscular atrophy observed in HSA-AR mice.

The functioning and composition of human platelet dense granules were studied using granules purified by Percoll fractionation. Reserpine-blockable serotonin (5-HT) uptake by the dense granule fraction was characterized and also demonstrated in the crude membrane fraction. Tetrabenazine (TBZ)-displaceable [3H]ketanserin binding was used to label the granular 5-HT transporter. Scatchard analyses, Hill plots, displacement curves, and binding kinetics indicated that TBZ-displaceable [3H]ketanserin binding labeled a site similar to that previously reported for chromaffin granules and synaptic vesicles. Analysis of phospholipid profiles in platelet fractions revealed that most platelet lysolecithin was associated with the dense granule fraction. Ganglioside analysis indicated that the predominant platelet ganglioside, GM3, was highly enriched in the dense granule fraction. The data lend further support to the idea that the 5-HT transporter complex is similar in platelet dense granules, chromaffin granules, and synaptic vesicles. However, the ganglioside and phospholipid findings clearly distinguish the types of storage sites and raise questions concerning the functional roles of dense granule GM3 and lysolecithin.

Testosterone and other androgens are thought to increase lean body mass and reduce fat body mass in men by activating the androgen receptor. However, the clinical potential of androgens for improving body composition is hampered by our limited understanding of the tissues and cells that promote such changes. Here we show that selective overexpression of androgen receptor in muscle cells (myocytes) of transgenic male rats both increases lean mass percentage and reduces fat mass. Similar changes in body composition are observed in human skeletal actin promoter driving expression of androgen receptor (HSA-AR) transgenic mice and result from acute testosterone treatment of transgenic female HSA-AR rats. These shifts in body composition in HSA-AR transgenic male rats are associated with hypertrophy of type IIb myofibers and decreased size of adipocytes. Metabolic analyses of transgenic males show higher activity of mitochondrial enzymes in skeletal muscle and increased O2 consumption by the rats. These results indicate that androgen signaling in myocytes not only increases muscle mass but also reduces fat body mass, likely via increases in oxidative metabolism.

1. No difference was observed in thein vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain. 2. In both cases there were progressive losses ofin vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals. 3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture. 4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers. 5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H+-ATPase. Unlike H+-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin Ai] and vanadate (IC50 100 μmol l−1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115×103, 39×103 and l6×103Mr), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an Mrof 63×103 instead of 67×103–70×I03; in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H+-ATPase, express the classical subunit A (70×103Mr). Moreover, two types of 57×103–60×103MrB subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.

Zebrafish is gaining popularity in behavioral neuroscience in general and in alcohol research in particular. Alcohol is known to affect numerous molecular mechanisms depending on dose and administration regimen. Prominent among these mechanisms are several neurotransmitter systems. Here we analyze the responses of the dopaminergic and serotoninergic neurotransmitter systems of zebrafish to acute alcohol treatment (1 h long exposure of adult fish to 0.00%, 0.25%, 0.50%, or 1.00% ethyl alcohol) by testing the concentration of dopamine, its metabolite DOPAC, and serotonin and its metabolite 5-HIAA from whole brain extracts. We utilize a sensitive HPLC method and describe significant alcohol induced changes in zebrafish for the first time. We show that dopamine significantly increased in a quasi-linear dose dependent manner, DOPAC showed a smaller apparent increase which was non-significant, while both serotonin and 5-HIAA showed a significant increase only in the highest acute dose group. We discuss the methodological novelty of our work and theorize about the implications of the neurotransmitter level changes from a behavioral perspective.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.

Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.

Fhe human body composition is assessed to determine percent body fat (PBF), fat mass (FM), and lean body mass or fat free mass (FFM). The topological distribution of body fat has been the subject of many studies in the world and India. To the best of our knowledge the present paper is the first report on the body composition in terms of PBF and FM, and their relationship with anthropometric measures in Muslim females in India. The present study examines anthropometric measurements and their relationship with the body composition among Muslim females of West Bengal, India. A cross-sectional study of 100 female, Muslim students of Howrah and Kolkata was undertaken to compare the relationships of biceps and triceps skinfold, waist, hip and upper arm circumference, waist hip ratio and conicity index with their body composition variables (PBF and FM). All anthropometric measures displayed significant (p < 0.05) correlation with body composition measures. The triceps skinfold, however, demonstrated a significant correlation with PBF (r = 0.90) and FM (r = 0.93). The greatest amount of variation of PBF (81.3 %) and FM (89.2 %) was explained by the triceps skinfold. In addition, a considerable amount of variation of PBF (72.8 %) and FM (86.0 %) was explained by the mid upper arm circumference. In conclusion, the present study displays a tendency of regional adiposity in the upper arm, triceps skinfold and mid upper arm circumference are much more strongly associated with body fat.

The present study investigated hormonal mediation of maternal behavior and accumbal dopamine (DA) responses to pup-stimuli, as measured in microdialysis samples collected from the nucleus accumbens shell of female rats in non-homecage environment. In Experiment 1, samples were collected before and after continuous homecage pup experience from either intact postpartum or cycling females. In Experiment 2, samples were collected before and after responding maternally in homecage from ovariectomized females given either parturient-like hormone or sham treatments. After baseline sample collection in the dialysis chamber, pup and food stimuli were individually presented to females. Upon sampling completion, all animals were placed back into their homecage with donor pups for several days, and then the sample collection procedure was repeated. Prior to stimulus presentation, postpartum and hormone-treated females had decreased basal DA release compared to their controls. In response to pup stimuli, only postpartum and hormone-treated females had increased DA release compared to basal release (both sampling days). In response to food stimuli, all females had increased DA responses from basal; although there were group differences on the initial day of sampling. Findings suggest that hormones associated with inducing maternal behavior in the postpartum rat play a significant role in modifying accumbal dopaminergic responses on first exposure to pup stimuli in the rat. However, the postpartum experience provides further modifications to this brain region to promote DA responses to pup stimuli.

Postpartum maternal experience produces long-lasting changes in maternal behavior in the mother rat, which can be altered by early-life isolation. Postpartum experience also affects the regulation of adult neurogenesis in the neural circuit underlying maternal behavior, in a region-specific manner. Female rats were reared either with their mothers (MR) or in isolation in an artificial rearing (AR) paradigm. In adulthood, rats were mated and separated from their pups at birth. The following day, dams were injected with a mitotic marker and either allowed to interact with pups (maternal experience) or left alone. Results show that MR rats that acquire a later maternal experience show increases in cell survival in parts of the excitatory limb of the maternal neural network (bed nucleus of the stria terminalis and nucleus accumbens), but no changes in the inhibitory limb (amygdala). In comparison to AR inexperienced rats, AR maternally experienced rats show no increases in cell survival in the excitatory limb, but a striking reduction in cell survival in the inhibitory limb. The results suggest that early preweaning maternal isolation alters the structural plasticity that occurs following a postpartum maternal experience.

The present study investigated the release of dopamine from the nucleus accumbens (shell) in response to pup-stimuli in the absence of lactation and maternal behaviors at time of sample collection. Subjects were female rats given maternal experiences through prior parturitions, recent pup-induced sensitization, or a combination of both. Nulliparous (N) or multiparous (M, had 2 prior litters but cycling) female rats either received pup-sensitization (S+) until they responded maternally in their homecage or no pup-sensitization (S-), thus, there were four groups: NS- (n=5), NS+ (n=6), MS- (n=5), and MS+ (n=8). Four hours after removal of pups (from homecage for S+ groups), all females were placed into the microdialysis chamber for sample collection. After baseline collection, four foster pups were given to the females. In this paradigm females show little to no maternal behavior in the test chamber. Samples (collected every 8 min) were analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) with electrochemical detection using HPLC. Relative to the inexperienced NS- females, the experienced NS+, MS- and MS+ females displayed significantly increased DA levels only during the first 8 min of pup-exposure. The more experience a female had with pups, the greater was the DA response (p.05). The results suggest that enhanced responding to pups following previous maternal experiences may be mediated through accumbal DA.

Rat pups reared apart from their siblings, mother, and nest environment in the 'pup-in-a-cup' regime show many alterations in behavior reminiscent of the Institutional Inattention/Overactivity Syndrome that characterizes children whose first few months are spent in institutions. In this report, we compare mother-reared (MR) and artificially reared (AR) male rats in concentrations and distributions of brain proteins that are involved in normal brain development. When assessed during the juvenile period and in adulthood, AR animals showed elevations in Neu-N (a neuronal marker) and in S-100 (an astrocyte marker) but reductions in synaptophysin (synapse protein), N-CAM (cell-adhesion molecule), GAP-43 (axon elongation protein), and BDNF (brain derived neurotrophic factor) in comparison to MR controls in many brain sites involved in attention, impulsivity, activity, and social behavior. Daily 'licking-like' stimulation provided to AR animals (AR-MAX) throughout early development that reverses many of the behavioral deficits, also reverses many of the isolation effects on brain proteins. Study 2 showed that elevations in the number of neurons in combination with decreases in functionality are associated with a reduction in neuronal pruning and apoptosis during the very early post-partum period in AR animals and their reversal through daily 'licking-like' stimulation.

Central body fat (CBF) as measured by waist circumference (WC) etc. has now been considered as risk factor for chronic disease, disability, and death. It has been assumed that one's characteristic level of body fat, as measured by body mass index (BMI), and CBF unfolds during the normal course of growth and development. However, studies of the development of CBF show relatively weak consistency in comparison to body fat. But to the best of our knowledge, no study in this aspect has been undertaken on any Indian population to study the effect of age and sex on body fat and CBF during development. In view of the above consideration, the present work has been undertaken among 179 Santal (a tribal population) children (Boys = 107, Girls = 72) of the Galudih area in East Singbhum, Jharkhand, India. Anthropometric measurements of height, weight, and circumference of waist were taken from each subject using standard procedure. The mean ages of boys and girls were 10.94 years and 9.48 years, respectively. The mean BMI for boys and girls were 14.48 kg/m2 and 13.78 kg/m2 Percent of variance explained by age was much greater for BMI (50%) as compared to WC (7-10%) in both sexes. Two way ANOVA showed that BMI had a significant (p0.05) effect of age and not of sex, whereas age-sex interaction had a significant impact on WC. The results of the present cross-sectional study indicated that during growth and development unfolding of CBF and BMI is independent of one another. Different set(s) of gene(s), onset of growth hormones during the normal development or their interaction could have been considered for this differentiation.

Cerebellar granular neurons were grown in culture up to 21 days and the protein compositions of undifferentiated (day 1), partially differentiated (day 7) and fully differentiated (day 21) neurons were analyzed by high-resolution 2D-gel electrophoresis. During neuronal differentiation there were not only increase in the amount of several known proteins, viz. actin, tubulin (both alpha and beta subunits), myosin (heavy and light chains), but very interesting changes were also observed in the expressions of different subunits and isoforms of those proteins. Furthermore, both in the acidic (pI 4.0-4.5) and alkaline (pI 7.0-8.5) regions interesting up and down regulations of several unidentified proteins were observed during the neuronal differentiation. These results indicated that there were several unidentified proteins that might be very valuable targets for studying regulation of neuronal differentiation. Research is going on for further characterization of those proteins using recently developed proteomics technology.

Sulfur mustard has been used as a vesicant chemical warfare agent. To understand the mechanism by which mustard gas exposure causes respiratory damage, we have used 2-chloroethyl ethyl sulfide (CEES) as a mustard analog. Our initial studies have shown that guinea pigs exposed to CEES intratracheally accumulate high levels of TNF-alpha. Accumulation of TNF-alpha leads to activation of both acid and neutral sphingomyelinases, resulting in high accumulation of ceramides, a second messenger involved in cell apoptosis. In addition, NF-kappa B was activated for a short period (1-2 h after exposure) as determined by mobility shift assay. Supershift assays indicated that both p50 and p65 of NF-kappa B were activated due to CEES exposure. However, NF-kappa B rapidly disappeared after 2 h. It is possible that the initial activation of NF-kappa B was an adaptive response to protect the cells from damage since NF-kappa B is known to inhibit TNF-alpha/ceramide-induced cell apoptosis. Since NF-kappa B disappeared after 2 h, the cells continued being damaged owing to accumulation of ceramides and activation of several caspases, leading to apoptosis.

Kallikreins are a group of specific serine proteases and are an integral part of kallikrein–kinin system. The kallikrein–kinin system is hypotensive in nature and counteracts with the renin–angiotensin system in the maintenance of normal blood pressure. So far, four kallikrein-like enzymes, namely, mK9, mK13, mK22, and mK26, have been known to convert the inactive pro-renin into biologically active renin. Some of these enzymes are induced by the thyroid hormone. In the proposed study, we investigated the effects of thyroid hormone on the expression of genes for mk9, mk13, and mk22 enzymes. We used guinea pigs as models because these animals share many characteristics in common to humans. Male adult guinea pigs were intramuscularly injected with 2 mg/kg body weight of thyronine. Forty-eight hours following the last injection, the liver was processed for Northern blot analysis using labeled mK9, mK13, and mK22 specific RNA probes. Only mK9 was found to be transcriptionally regulated by the hormone.

We have demonstrated earlier that thyroid hormone (T3) regulates the activity of cholinephosphotransferase (CPT) in guinea pig lung. This effect of T3 is not organ specific because we found T3 also regulates CPT activity in the guinea pig liver. Northern blot analysis using two oligonucleotide probes, one synthesized on the basis of the yeast CPT gene sequence and another on the basis of partial cDNA clone from guinea pig CPT clone, revealed that T3 stimulates the expression of new CPT mRNA. Studies with transcriptional and translational inhibitors indicated that T3 enhanced the translation of the CPT mRNA as well as translocation of preformed CPT enzyme protein from cytosol to mitochondria. Furthermore, it strengthens our previous finding that yeast CPT and guinea pig CPT have high homology in their sequence as both the oligonucleotide probes gave the similar type of Northern blot in the present study.

Bone resorption, a process necessary for bone growth and remodeling as well as bone repair, is performed by the multinucleated osteoclast. Abnormal regulation of bone resorption is the cause of several clinical entities. Osteoclastic bone resorption can be increased and lead to local or systemic bone loss (bone tumors, osteoporosis, osteoarthritis) or genetically deficient as in various types of osteopetrosis.

Neuro-2a neuroblastoma cells, when differentiated via a cAMP-dependent pathway by treatment with anti-GM 3 monoclonal antibody, accumulated a high level of pp60 c-src protein and pp60 c-src kinase activity just before the onset of neurite formation. The specific kinase activity of the accumulated c-src protein was found to be comparable to that of normal cerebellar neurons, but was about 6- to 8-fold higher than that of normal astrocytes. These results, and migrations of peptide fragments in the SDS-polyacrylamide gels after V8 proteolysis, strongly indicate the accumulation of the neuron-specific isoform of the c-src protein (pp60 c-srcN ) in the GM 3 antibody-treated Neuro-2a cells. Similar high levels of pp60 c-src protein and pp60 c-src kinase activity were observed in the Neuro-2a cells differentiated via a cAMP-dependent pathway by treatment with dibutyryl cAMP, but not in the same cell line when differentiated via a cAMP-independent pathway with 5-bromo-2′-deoxyuridine. These results demonstrate that the accumulation of high levels of the neuron-specific isoform of the pp60 c-src protein (pp60 c-srcN ) in the Neuro-2a neuroblastoma cells depends on the specific signal transduction pathway involved during the differentiation of these cells.

The osteoclast proton pump (OC H(+)-ATPase) differs from other vacuolar H(+)-ATPases (V-ATPases) in its sensitivity to vanadate and in the subunit composition of its catalytic domain, where isoforms of subunits A and B are expressed [Chatterjee et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6257-6261]. In the present study, the sensitivity of the osteoclast H(+)-ATPase to various oxyanions was tested. The results indicate that H+ transport by microsomal preparations isolated from chicken osteoclasts is 20-100-fold more sensitive to nitrate that any other animal and fungal V-ATPases and 10-20-fold more sensitive than plant V-ATPases, as is the ATPase activity of the affinity-purified enzyme. This inhibition by nitrate is not due to a chaotropic effect of the oxyanion and is complete at 1 mM concentrations with an IC50 of 100 microM. In contrast, proton transport by the OC H(+)-ATPase was insensitive to other oxyanions (phosphate, sulfate, and acetate) which inhibit other V-ATPases. These results further demonstrate that the proton pump present in osteoclast membranes differs from other vacuolar ATPases. It is speculated that, since cells of the macrophage lineage can generate high intracellular concentrations of nitrate, it may be possible to physiologically or therapeutically regulate the activity of the OC H(+)-ATPase in the osteoclast without affecting the other V-ATPases in the same or in other cells.

Publisher Summary This chapter discusses the cellular and molecular biology of the osteoclast. The osteoclast is the cell responsible for the resorption of the bone matrix. Bone resorption is a necessary process for the normal development of the skeleton, for its adaptability, and for its maintenance. This cellular process is essential in the growth, remodeling, and repair of bone and is, under normal conditions, tightly coupled to the process of bone formation by the osteoblast. It is the balance between these two cellular activities that determines skeletal mass and shape at any point in time. The main determinants of the biology of the osteoclast are first its attachment to the bone matrix, leading to the formation of the sealed-off bone resorbing compartment and, second, the polarized acidification of, and secretion of enzymes into, this compartment. These activities require tight control of different membrane domains both in terms of their composition and targeting and in terms of ion transport. The biology and activity of these membrane domains are strongly interdependent.

The osteoclast is the cell responsible for the resorption of the extracellular bone matrix. Under physiological conditions, bone resorption plays an essential role in the homeostasis of both the skeleton and serum calcium. This cellular process is also essential in the growth and remodeling of bone, where it is tightly coupled to the process of bone formation by the osteoblast. It is the integrated functions of these two cell types that lead to the quantitative and qualitative maintenance of the skeleton, to the changes in size and shape of the individual bones during growth, and to bone repair after trauma or fracture. On the other hand, it is the disruption of the coupling between bone resorption and formation that leads to abnormally dense (osteopetrosis and osteosclerosis) or porous bone (osteoporosis). A coupled but high resorption rate characterizes high bone-turnover diseases, such as hyperparathyroidism or Paget’s disease, and leads to the disruption of the architecture and function of the skeleton.

Analysis of proton (H+) transport by inside-out vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (N-ethylmaleimide and Bafilomycin A1) and vanadate (IC50, 100 microM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of 57- to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class of H+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.

A monoclonal antibody against GM3 ganglioside (GM3 Ab) was found to trigger differentiation of Neuro-2a cells in culture. The differentiation of Neuro-2a cells by GM3Ab was accompanied by increased levels of intracellular serotonin and amino acid neurotransmitters viz. aspartate, glutamate, glutamine, glycine and taurine. Further study indicated that the increase in the serotonin level was not due to a higher rate of serotonin synthesis but rather to a higher rate of active transport of serotonin from the medium. Studies on the cell surface gangliosides revealed that unlike the proliferating cells, the GM3Ab-mediated differentiated cells contained higher gangliosides in addition to GM3 and GM2 gangliosides. Analysis of total cellular proteins indicated the appearance of a 25 kDa protein, pI 5.4, in the GM3Ab-treated cells—a small amount of this protein was observed in dibutyryl cAMP (Bt2cAMP)-treated cells, however, the protein was totally absent in the 5-bromo-2′-de-oxyuridine (BrdU)-treated cells. Investigation of the mode of action of GM3Ab indicated that the cellular differentiation was due to increased cAMP accumulation resulting from an increase in the adenylate cyclase activity. Further studies with different agents affecting protein kinase C (PKC) activity and direct assay of PKC ruled out the possibility that GM3Ab mediated its effect via PKC. This GM3Ab-induced differentiation could be inhibited by protein kinase A (PKA) inhibitor, H8, but could not be inhibited by sphingosine, an inhibitor of PKC. Pertussis toxin could mimic the effect of GM3Ab, suggesting that GM3Ab caused the elevation in the adenylate cyclase activity by reducing the Gi-protein inhibition of the adenylate cyclase. The data suggests that GM3Ab, after interaction with cell surface GM3, elevated intracellular cAMP level by withdrawing the inhibitory effect of some undefined factor(s) present in culture medium which normally keeps adenylate cyclase activity low through activation of Gi-protein.

An improved method for the isolation of human platelet dense granules was developed. A good yield (45%) of highly enriched (69-fold, based on serotonin content) dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of Km and Vmax values for [3H]-serotonin uptake, as well as the first determination of Kd and Bmax values for tetrabenazine-displaceable [3H]-ketanserin binding, in the human platelet dense granule. The rates and affinities (Vmax 1.45 nmol/mg/min, Km 0.93 uM) of [3H]-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable [3H]-ketanserin binding was observed with a Kd (9.4 nM) similar to, and a Bmax (5.4 pmol/mg) approximately 10-fold lower than, that previously seen in bovine chromaffin granules.

The planned and ongoing studies of platelet function and composition should allow us to better define the alteration which we presume to be present in platelets of autistic subjects. Although much of the research focuses on serotonergic aspects, the more general research should permit a better delineation of the extent of the alteration and will protect against a premature narrowing of the inquiry. The methodological development which has been a necessary aspect of the work should contribute to an improved understanding of platelet function and composition, as well as result in improved clinical tools for the assessment of platelet functioning in neuropsychiatric disorders and hematology. As an example, improvements in short-term in vitro storage conditions to stabilize aggregation and shape change responses over time were found to be necessary, and are probably critical to an optimal comparison of these phenomena across groups. The identification of the platelet alteration which is responsible for the hyperserotonemia of autism should prove useful in several ways. It would be expected that assessment of the altered function would provide a marker with less overlap with the normal population than the multidetermined measure of blood 5-HT. Determination of the specific protein(s) involved in the altered platelet should lead directly to gene probes and chromosomal location. These, in turn, should prove useful for neonatal screening, subtyping, and more powerful genetic and family studies. Work of this sort might also allow early intervention and improved treatment. Finally, characterization of the physiological alteration would provide a basis for focusing studies of brain neurochemistry and should, as well, suggest modes of neuropharmacological intervention. The confidence that one can have in the basic finding of hyperserotonemia in autism and the potential benefits to be derived from its explication make further research in this area of great interest.

To the Editor. — We have measured levels of monoamines, monoamine metabolites, amino acids, and neurotransmitter-related enzymes in subcortical regions of postmortem tissue obtained from brains of four individuals who had Gilles de la Tourette's syndrome (TS). Although the study was limited by the small number of available brains and other concomitants of postmortem analyses, several suggestive differences were seen between the brains of subjects with TS and those of the control group. Most intriguing in terms of possible pathophysiologic significance were the observation of generally lower values of serotonin and its related compounds in the TS group, and the lower TS group means of glutamate in the three major projection areas of the subthalamic nucleus (STN): the medial globus pallidus (MGP), lateral globus pallidus (LGP), and substantia nigra reticulata (SNR). Subjects and Methods.— The brains of four subjects who were diagnosed before death as having TS were obtained. One

Partial purification of the dihydropyridine receptor from rat skeletal muscle demonstrated mainly a 60 kDa band in SDS-polyacrylamide gel. An antibody raised against that protein behaved as a calcium channel agonist viz. Bay K8644. The affinity purified antibody, when added to cultured heart cells, increased the beat rate 40–80% depending on the titer of the antiserum. The antibody also woke up the beats of the cells previously blocked with the channel antagonist, nifedipine. Immunoblot analysis indicated that the receptor of this antibody in heart cell membrane is also a 60 kDa protein.