Microanalysis of free and conjugated bile acids by thin-layer chromatography and in situ spectrofluorimetry

A combination of thin-layer chromatography (TLC) and in situ spectrofluorimetry for the determination of free bile acids and bile acids conjugated with glycine or taurine is described. This method makes it possible to determine bile acids concentrations as low as 0.15-0.25 nmol (0.05-0.1 microgram) in a simple and reproducible way. Moreover, information can be obtained about conjugation patterns and relative concentrations of mono-, di-, and trihydroxy bile acids as well as about the presence of abnormal bile acids. After TLC the bile acids are made visible in uv light by dipping the layer in sulfuric acid in diethyl ether and warming it under well-described conditions. The fluorescence of the bile acids on the thin layer can be measured and makes it possible to quantitate them. The method presented here is applicable to bile acid-containing extracts from serum, bile, and feces, and the results are in good agreement with those obtained by enzymatic and gas-liquid chromatographic techniques.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

Extravascular circulation in the pituitary of Mugil cephalus (Teleostei)

Summary Extravascular circulation in the pituitary of Mugil cephalus was investigated by injecting live fish with horseradish peroxidase and studying the distribution of the enzyme in the gland. The principal components of the extravascular circulatory system are the pericapillary spaces, and, arising from them, the interlobular and circumhypophyseal spaces. Extensions of these spaces penetrate the glandular parenchyma of the pars distalis, where they merge with pericellular spaces. In the neurohypophysis, pericapillary spaces are connected to the periaxonal spaces.Capillaries penetrating from the proximal neurohypophysis into the pars distalis are accompanied by neurosecretory axons. These axons form a mass of tissue which is limited near the capillaries by the pericapillary spaces and near the adenohypophysis by the interlobular spaces. Toward the interior of the adenohypophysis the amount of nervous tissue accompanying the capillaries progressively diminishes, thus reducing the distance between pericapillary and interlobular spaces. Within the pars distalis, the neurosecretory axons accompanying the capillaries are sparse, and the secretory and stellate cells are mostly located directly adjacent to the pericapillary spaces. In the neuro-intermediate lobe, interlobular spaces outline the neuro-adenohypophyseal boundary.The relationship between extravascular spaces and hormone-secreting cells varies in the different regions of the adenohypophysis depending upon the type of neurosecretory innervation in the respective region. In the regions of prolactin and gonadotropin cells, where neurosecretory axons are in direct contact with the secretory cells, the hormone-secreting and stellate cells are adjacent to the pericapillary spaces. In the regions of ACTH and STH cells, secretory and stellate cells are found adjacent to the interlobular spaces, which are interposed between the cells and the neurosecretory axons.Abbreviations AH adenohypophysis - CH circumhypophyseal - DNH distal neurohypophysis - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - PD pars distalis - PI pars intermedia - PPD proximal pars distalis - RNH rostral neurohypophysis - RPD rostral pars distalis This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Evidence for restricted oligosaccharide mobility at the erythrocyte membrane surface: A fluorescence study

The glycophorins of whole, human erythrocytes were labeled at their sialic acid residues with one of three fluorescent probes. After preparation of the erythrocyte ghosts, the mobility of each fluorescent probe on the intact membrane was compared with its mobility on the isolated, labeled glycopeptides dissolved in aqueous buffer. A four- to ninefold decrease in the rotational relaxation time, as defined by the Perrin equation, accompanied the proteolytic removal of the labeled glycopeptides from the membrane. This suggests that the fluorescent probes, and by extrapolation, the sugars to which they are immediately attached, are restricted in their mobility at the membrane surface. A crude model of the carbohydrate layer of the erythrocyte surface was constructed by incorporating the labeled, tryptic glycopeptides into agarose gels of different agarose content. A decrease in the probe's mobility was observed as agarose content was raised. This indicates that the high oligosaccharide density at the erythrocyte membrane surface may contribute to the observed immobilization of the fluorescent probes in situ.     

Crystallization of the receptor binding domain of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the product on of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2₁, with cell parameters a = 55.6 Å, b = 60.4 Å, c = 77.7 Å, β = 90.0°, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF. © 1996 Wiley-Liss, Inc.     

Purification,partial characterization,and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes,spermatids, and sperm

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm-associated keratin) from sodium dodecyl sulfate-β-mercaptoethanol(SDS-βME)-dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15-mer and 10-mer fragments with 70–100% homology to human, rat, and mouse keratins and corresponding to the 1A and 2A regions of the α-helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10-mer amino acid sequence KAQYEDIAQK (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti-MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunobloting, and [³²P]orthophosphate-labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9–6.3, regarded as a precursor, and both detergent-insoluble and soluble 57 kDa protein with pl range 5.0–5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent-insoluble form and its pl was more acidic (4.7–4.8). Whole-mount electron microscopy of negatively stained preparations of sperm-derived Sak57 resuspended in SDS-βME revealed a rod-shaped pattern. A decrease in the concentration of SDS-βME resulted in the side-by-side aggregation of rod-shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three-dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10–11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis. © 1996 Wiley-Liss, Inc.     

The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

Calcium regulation of senescence in rose petals

Rose plants grown at high relative humidity (RH) produce flowers with a shorter vase life than those grown at low RH. The calcium content of the former is lower than that of the latter. The present study was conducted to examine the possible involvement of calcium in the regulation of rose flower senescence. In whole cut flowers and in detached petals of cvs Mercedes and Baroness, CaCl₂ treatment promoted bud-opening and delayed senescence. The treated flowers stayed turgid and continued their initial postharvest growth for longer periods of time. The membrane protein content in detached petals decreased with time, in parallel to the decline in membrane phospholipids (PLs). Calcium treatment delayed the decrease in both membrane proteins and PL and increased ATPase activity in the aging petals. Electrolyte leakage, which is a reliable indicator of petal-membrane senescence, was postponed in calcium-treated flowers. Calcium treatments also sukppressed ethylene production with age. We suggest that the calcium-induced delay in rose petal senescence involves the protection of membrane proteins and PLs from degradation, thus preserving the integrity of the membranes, reducing ethylene production, and hence maintaining solute transport and tissue vitality.