Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones a-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation. (C) 2001 Elsevier Science B.V. All rights reserved.

Peripheral T-Cell tolerance defined through transgenic mouse studies

Selection in the thymus restricted by MHC and self-peptide shapes the diverse reactivities of the T-cell population which subsequently seeds into the peripheral tissues, in anticipation of the universe of pathogen antigens to which the organism may be exposed. A necessary corollary is the potential for T-cell self-reactivity (autoimmunity) in the periphery. Transgenic mouse models in which transgene expression in the thymus is prevented or excluded, have been particularly useful for determining the immunological outcome when T-cells encounter transgene-encoded 'self' antigen in peripheral tissues. Data suggest that non-mutually exclusive mechanisms of T-cells 'ignoring' self-antigen, T-cell deletion, T-cell anergy and T-cell immunoregulation have evolved to prevent self-reactivity while maintaining T-cell diversity. The peripheral T-cell repertoire, far from being static following maturation through the thymus, is in a dynamic stated determined by these peripheral selective and immunoregulatory influences. This article reviews the evidence with particular reference to CD8+ive T-cells.

Timing and magnitude of peak height velocity and peak tissue velocities for early, average, and late maturing boys and girls

Height, weight, and tissue accrual were determined in 60 male and 53 female adolescents measured annually over six years using standard anthropometry and dual-energy X-ray absorptiometry (DXA). Annual velocities were derived, and the ages and magnitudes of peak height and peak tissue velocities were determined using a cubic spline fit to individual data. Individuals were rank ordered on the basis of sex and age at peak height velocity (PHV) and then divided into quartiles: early (lowest quartile), average (middle two quartiles), and late (highest quartile) maturers. Sex- and maturity-related comparisons in ages and magnitudes of peak height and peak tissue velocities were made. Males reached peak velocities significantly later than females for all tissues and had significantly greater magnitudes at peak. The age at PHV was negatively correlated with the magnitude of PHV in both sexes. At a similar maturity point (age at PHV) there were no differences in weight or fat mass among maturity groups in both sexes. Late maturing males, however, accrued more bone mineral and lean mass and were taller at the age of PHV compared to early maturers. Thus, maturational status (early, average, or late maturity) as indicated by age at PHV is inversely related to the magnitude and late maturers for weight and fat mass in boys and girls. Am. J. Hum. Biol. 13:1-8, 2001. (C) 2001 Wiley-Liss, Inc.

Relationships between crude protein and mineral concentrations in alfalfa and value of alfalfa silage as a mineral source for periparturient cows

One hundred and eight samples from three cultivars of alfalfa were obtained from three cuttings in 1996-1998 to evaluate the relationship between crude protein (CP) and mineral concentrations of alfalfa with cutting and maturation. The CP content drastically decreased from 27.9 to 11.4% on DM with maturity. Highly positive correlations were observed between CP and K in the first and the second cutting of alfalfa. The Ca content remained almost constant throughout the growth period. Four multiparous Holstein cows were assigned an alfalfa silage diet or an orchardgrass silage diet from 3 weeks prepartum to 1 week postpartum to examine the effect on the mineral balance. In the prepartum and postpartum diet, the roughage to concentrate ratio was 70:30 and 50:50, with alfalfa being 50 and 100% of the roughage, respectively. The alfalfa contained 1.93% of K. No metabolic disorders occurred, but the body weight decreased drastically from 1 to 6 days postpartum with each diet because of the high milk production immediately after the parturition. Positive retention of N, Ca, P, Mg, and K was observed prepartum, whereas the cows had negative N and mineral retention from 2 to 4 days postpartum. The Ca and P absorption, and Mg retention of cows with the alfalfa diet were higher than with the grass diet. The plasma Ca and inorganic P were not affected by diet, but the plasma PTH at parturition and plasma hydroxyproline from 1 week prepartum to 1 week postpartum were higher with the alfalfa diet. These results suggest that the low K alfalfa is suitable not only to prevent the incidence of milk fever but also to increase Ca, P and Mg utilization of periparturient cows, but the mineral supplementation is needed for the postpartum cows immediately after the parturition. (C) 2001 Elsevier Science B.V. All rights reserved.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Relationship between endosomes and lysosomes

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy Studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

Inhibition of adventitious rooting in Backhousia citriodora F. Muell cuttings correlate with the concentration of essential oil

Backhousia citriodora is typical of the many commercially valuable woody Australian Myrtaceae species that are recalcitrant in forming adventitious roots from cuttings after maturation. A series of experiments were conducted to identify an endogenous rooting inhibitor in line with established criteria. Endogenous levels of citral were correlated with the rooting capacities of juvenile versus mature, and easy- versus difficult-to-root genotypes of B. citriodora, in both winter and summer. The biological activity of citral was confirmed in bioassays on mung beans and easy-to-root B. citriodora seedlings. Evidence of a common mechanism of root inhibition with other species in the Myrtaceae and the role of action of citral are discussed.

Long-lasting synaptic modification in the rat hippocampus resulting from NMDA receptor blockade during development

Recent reports have suggested that proper maturation of synapses in the hippocampus requires activation of NMDA receptors. We previously demonstrated that neonatal ethanol exposure results in a lasting reduction in synaptic strength in the hippocampus. To determine if this reduction was due to ethanol's effects on NMDA receptors, we investigated long-term changes in synaptic properties resulting from administration of NMDA receptor antagonists to neonatal animals. Rats were injected daily from PND 4-9 with either the noncompetitive NMDA receptor antagonist MK-801, the competitive NMDA receptor antagonist CPP, or the AMPA receptor antagonist NBQX. Control rats were either injected daily with physiological saline during the same period or left to develop normally. Hippocampal slices were prepared from nembutal-anesthetized animals between PND 35 and PND 40. The maximum pEPSP and PS values were not significantly different between controls and NMDA antagonist-treated animals. However, slices from animals injected with NMDA receptor antagonists required higher stimulus currents to attain comparable pEPSPs. The ratio of the slope of the pEPSP to the amplitude of the presynaptic volley was also reduced, as were pEPSP responses to specific stimulus currents. None of these effects were observed in slices prepared from animals treated with the AMPA receptor antagonist NBQX. Glutamate receptor antagonism did not produce lasting changes in long-term potentiation or paired-pulse facilitation. These results indicate activation of NMDA receptors during development is necessary for proper development of synapses. (C) 2001 Wiley-Liss, Inc.

Towards the clonal propagation of coconut

Past studies from our laboratory have shown that whole immature, or mature sliced, zygotic embryos are a very good starting explant for coconut somatic embryogenesis. The highest rate of somatic embryogenesis was obtained when certain polyamines were added into the culture medium as well as activated charcoal (AC) to absorb unwanted phenolics. These past studies also showed that the development and maturation of the somatic embryos produced could be improved by the addition of abscisic acid (ABA), alone or with one of several osmotically active agents, into the culture medium. In the present study this well characterised somatic embryogenic system for zygotic tissues is being modified and applied to somatic tissues. This recent approach should be a better method for the rapid production of clonal, true-to-type coconut palms. The present research approach is focused on young leaf section explants which have been found to be very responsive to callus production. Young leaf sections produced optimum callus when cultured on media containing 2,4-D (150 μM) and the amount produced could be increased by soaking the sections in sterile water (15 to 60 minutes) or ascorbic acid (15 to 30 minutes) prior to culturing. Further improvement in callus production, as well as a reduction in the time taken for callogenesis was obtained when casein hydrolysate and/or certain polyamines were added to the callus induction medium. The development of the somatic embryos was improved by using ABA and polyethylene glycol (PEG) in the maturation medium. Despite these initial successes in improving coconut somatic embryogenesis, further studies are now being considered to shorten the time to achieve somatic embryogenesis, to better germinate somatic embryos and to improve the rate of somatic seedling conversion into plantlets.

Expression of neurexin ligands, the neuroligins and the neurexophilins, in the developing and adult rodent olfactory bulb

The neurexins are a large family of neuronal cell-surface proteins believed to be involved in intercellular signalling and the formation of intercellular junctions. To begin to assess the role of these proteins in the olfactory bulb, we describe here the expression patterns of their transmembrane and secreted ligands, the neuroligins and neurexophilins, during both embryonic and postnatal development. In situ hybridisation showed that neuroligin 1 and 2 were expressed by second order mitral cells during early postnatal development but not in adults. The secreted ligand for a-neurexin, neurexophilin 1, was also expressed in the postnatal olfactory bulb. Neurexophilin 1 was detected in only periglomerular cells during the early postnatal period of glomerular formation but later was also expressed in mitral cells. These results suggest that neurexin-ligand interactions may be important for development and/or maturation of synaptic connections in the primary olfactory pathway.

Structure of CcmG/DsbE at 1.14 angstrom resolution: High-fidelity reducing activity in an indiscriminately oxidizing environment

CcmG is unlike other periplasmic thioredoxin (TRX)like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.

Induction of birth in the bandicoot (Isoodon macrourus) with prostaglandin and oxytocin

As in eutherians, maturation of the fetal pituitary and adrenal glands together with an increase in prostaglandin and mesotocin or oxytocin production initiates birth in marsupials. in this study, prostaglandin (Lutalyse) or oxytocin (Syntocinon) were administered to pregnant bandicoots at 05:00 h on the calculated day of birth and the resultant effects were filmed for analysis. The administration of prostaglandin caused the bandicoot to adopt the birth position several minutes after injection (n = 2). However, the bandicoot did not give birth for several hours. Birth occurred at a similar time of day to that observed for untreated bandicoots (n = 7), between 08:00 h and 12:00 h. After an injection of oxytocin, the bandicoot assumed the birth position and birth occurred within several minutes. The young were alive while still connected to their allantoic stalks. However, they were unable to attach to the teats and did not survive (n = 4). The induced young were the colour of venous blood and died soon after the umbilicus was separated, indicating that the cardiopulmonary system of these neonates was underdeveloped and inadequate to maintain life. The results from this study demonstrate that prostaglandin is required to prepare the bandicoot for birth, and mesotocin is required for contraction of the uterus and for birth to occur.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

Responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on aortae from normo-, pre and hypertensive rats

The objective of this study was to determine the responsiveness, affinity constants and beta-adrenoceptor reserves for isoprenaline on the isolated aorta in the maturation of normotensive and hypertensive rats. The effects of a very slowly reversible antagonist, bromoacetylalprenololmenthane (BAAM), on the relaxant responses of the aortae of 5- and 14-week-old Wistar Kyoto normotensive rats (WKY) and spontaneously hypertensive rats (SHRs) to isoprenaline were determined. Five-week-old SHRs are pre-hypertensive and the aortic rings are less responsive to isoprenaline than age-matched WKY (pD(2) values: WKY, 8.40; SHRs, 8.03). Similar relaxant responses to forskolin were obtained on the aortae of 5- and 14-week-old WKY and SHRs. The K-A value for isoprenaline at the aortic beta(2)-adrenoceptors of the 5-week-old WKY was 2.1 x 10(-7) M, and similar values were obtained on the aortae of 5-week-old SHR and 14-week-old WKY and SHRs. In the maturation of the WKY aortae from 5 to 14 weeks, there was a reduction in the maximum response, a major loss of sensitivity and a loss of 2-adrenoceptor reserve for isoprenaline. On 5-week-old SHR aorta, the sensitivity to isoprenaline was 2.5-fold lower, and the beta(2)-adrenoceptor reserve was less than on age-matched WKY. In the development of hypertension on the SHR aorta from 5 to 14 weeks, there was a reduction in the maximum response to isoprenaline. At 14 weeks, the sensitivity and the 2-adrenoceptor reserve to isoprenaline were similar, but the maximum responses were lower on the SHR than WKY. As there are differences in pre-hypertensive SHR and age-matched WKY aortic responses to isoprenaline, it is no longer valid to consider that the loss of responsiveness to isoprenaline in hypertension is solely owing to the hypertension. There are no changes in affinity, but major changes in the sensitivity, maximum responses and aortic beta(2)-adrenoceptor reserves to isoprenaline in the maturation of normotensive and pre-hypertensive aortae.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.