Proteomic analysis of primary cultures of human adipose-derived stem cells: modulation by Adipogenesis

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by > or = 2-fold, whereas 13 showed a > or = 3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin alpha phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.     

Human sperm proteins from testicular and epididymal origin that participate in fertilization: modulation of sperm binding to zona-free hamster oocytes, using monoclonal antibodies.

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme-linked immunosorbent assay (ELISA). Immunoperoxidase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the beginning to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona-free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation.     

Grasping the Changes Seen in Older Adults When Reaching for Objects of Varied Texture

Old age is associated with reduced mobility of the hand. To investigate age related decline when reaching-to-lift an object we used sophisticated kinematic apparatus to record reaches carried out by healthy older and younger participants. Three objects of different widths were placed at three different distances, with objects having either a high or low friction surface (i.e. rough or slippery). Older participants showed quantitative differences to their younger counterparts – movements were slower and peak speed did not scale with object distance. There were also qualitative differences with older adults showing a greater propensity to stop the hand and adjust finger position before lifting objects. The older participants particularly struggled to lift wide slippery objects, apparently due to an inability to manipulate their grasp to provide the level of precision necessary to functionally enclose the object. These data shed light on the nature of age related changes in reaching-to-grasp movements and establish a powerful technique for exploring how different product designs will impact on prehensile behavior.     

Localization of lipoprotein unesterified cholesterol in nondenaturing gradient gels with filipin

A method is described for the staining of lipoprotein unesterified cholesterol in nondenaturing polyacrylamide gradient gels with the fluorescent polyene antibiotic, filipin. The sensitivity of the filipin stain was comparable to that of oil red O and Coomassie R250 in terms of the amount of lipoprotein applied. Filipin successfully stained discoidal complexes of apoA-I-phosphatidylcholine-cholesterol, which in turn were stained poorly with oil red O. The potential for the identification of unesterified cholesterol-enriched lipoprotein subclasses was demonstrated.     

Genetic duplication in the white-split interval of the X chromosome in Drosophila melanogaster

A detailed cytogenetic study of male-viable and lethal deficiencies affecting the w-spl interval in Drosophila melanogaster has revealed the existence of genetic duplication such that, for example, the consequences of the loss of salivary chromosome band 3C3 are essentially compensated for by the presence of band 3C5-6, and vice versa. Although each of the duplicate elements possesses rst ⁺ and vt ⁺ activity, rst and vt phenotypes appear in males when 3C3 and part, but not all, of 3C5-6 are deleted. The degree of rst and vt expression can be correlated with the amount of material lost from 3C5-6. Deletions removing the entire 3C3-6 interval are male lethal. Despite the duplicate elements, at least one EMS-induced, presumptive point mutation expressing only rst is known; two others express both rst and vt. No loci other than rst and vt occur between W and spl. Band 3C2 appears to be associated with the w locus, which probably extends into the interband space between 3C1 and 3C2. The w locus is not involved in the rst-vt duplication in the 3C3-6 region. — The cytogenetic characteristics of the 3C region—a high coefficient of crossing over, frequent induced chromosome breakage, ectopic pairing, constriction, and an extended replication period—can be correlated with the fact that in 3C a relatively long stretch of DNA, nearly 2% of the entire X chromosome, is highly compacted into but few adjacent bands. These characteristics do not necessarily represent special properties of intercalary heterochromatin; they can be interpreted as reflecting the properties of any similarly organized euchromatic region.This investigation was aided by research grants from the U. S. Public Health Service (GM 13631) to G. Lefevre, Jr. and the National Science Foundation (GB 27599) to M. M. Green.