Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Variation in plasma constituents during the natural vitellogenic cycle of tuatara,Sphenodon Punctatus

• 1.1. The vitellogenic cycle of female tuatara was investigated by monitoring plasma levels of vitellogenin, calcium, total protein, inorganic phosphate (P₁) and cholesterol.
• 2.2. Vitellogenin was not detected in females in the non-reproductive condition, but was found perenially in plasma of reproducing females during vitellogenesis, which normally lasts about 3 years out of the 4 year ovarian cycle.
• 3.3. No large year-to-year variations were found in the plasma constituents measured and there was no correlation between the oestradiol peak at mating and plasma levels of vitellogenin.
• 4.4. The results provide further evidence that tuatara have an extraordinary prolonged and gradual vitellogenic cycle spanning several years for a single clutch of eggs. This type of reproductive cycle is unique among reptiles.

     

Specificity of transcription of single-copy DNA in different rat tissues

• 1.1. The amount of single-copy DNA sequences transcribed in normal tissues of adult rats (brain and liver) and in the Guerin ascites tumor (GAT) was determined by hybridization of in vitro labelled ¹²⁵I-single-copy rat DNA with a vast excess of total nuclear RNA to very high Rot values (up to 350,000).
• 2.2. The tissue specificity of total nuclear RNA (nRNA) was estimated by annealing of single-copy DNA to a mixture of nuclear RNAs of two different organs (brain + GAT; liver + GAT).
• 3.3. Liver and GAT RNAs annealed to about 12% of the single-copy DNA.
• 4.4. Hybridization with a mixture of the two RNAs increased slightly the amount of hybridization.
• 5.5. In contrast to other tissues, brain nuclear RNA hybridized to a much higher level (20% of the single copy DNA). Addition of GAT RNA did not increase this value.

     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

Age-related changes in total dna and rna and incorporation of uridine and thymidine in rat liver,kidney and spleen

• 1.1. Total content of DNA and RNA in liver, kidney and spleen were measured in young and aged rats. At the same time the incorporation of [¹⁴C]thymidine, a DNA precursor, and [³H]uridine, an RNA precursor, were also determined.
• 2.2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporation of precursors.
• 3.3. Young animals have more DNA per organ relative to RNA. with kidney and spleen DNA showing a decrease between maturity and senescence.
• 4.4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [³H]uridine uptake decreases.
• 5.5. Liver polyploid differentiation, and [¹⁴C]thymidine and [³H]uridine uptake, are correlated.
• 6.6. In kidney, incorporation of [³H]uridine is inversely related to [¹⁴C]thymidine incorporation.

     

Comparative study of three different forms of both native and subunit vitellogenin identified in quail plasma

• 1.1. Three different species of native vitellogenin, designated Vgα, Vgβ and Vgγ, were detected by gradient polyacrylamide gel electrophoresis (2–16%) in the plasma of untreated mature female quail and in the plasma of estrogen-induced female or male quail. The molecular weights of Vgγ, Vgβ and Vgα (in order of increasing size) were estimated to be 400,000 to 450,000 (by PAGE) or 466,000 (by analytical ultracentrifugation).
• 2.2. DEAE-cellulose chromatography resolved vitellogenin-containing plasma into peak IV, fractions of which contained Vgα and Vgβ in equal quantities, and peak III, fractions of which contained Vgα, Vgβ and Vgγ in varying proportions.
• 3.3. Peak IV fractions disssociated to give two bands (designated Vg1 and Vg2) on SDS-polyacrylamide gel electrophoresis. Pooled peak III fractions and plasma from untreated female or estrogen-induced female and male quail dissociated to give three bands (Vg1, Vg2 and Vg3). The mol. wt of Vg1, Vg2 and Vg3 were approx. 232,500, 212,000 and 194,000, respectively.
• 4.4. Peak III and peak IV vitellogenin fractions were shown to have similar amino acid compositions except that the peak III vitellogenin fraction contained twice as much cystine as the peak IV vitellogenin fraction (2.2 vs 1.1 mol%). The peak IV vitellogenin fraction contained more serine than the peak III vitellogenin fraction (11.8 vs 10.9 mol%) and more phosphorus (0.584 vs 0.516 nmol/μg protein).
• 5.5. Vgα or Vg1, in trace amounts, were detected in the plasma of untreated male quail.
• 6.6. The amino acid contents, phosphorus contents, and mol. wt of quail vitellogenins were similar to published values for other egg-laying species.

     

Kinetic studies of vitellogenin metabolism in the elasmobranch Scyliorhinus canicula L.

• 1.1. The appearance of vitellogenin in the plasma of adult female Scyliorhinus canicula and its subsequent disappearance and conversion into yolk granules were monitored by an isotopic method. Rates of vitellogenin synthesis in different fish were compared.
• 2.2. There was considerable individual variation in the rate of synthesis and in the plasma half-life of vitellogenin; measured values of the latter ranged from 132 to 303 hr (mean 216 hr) at 7 ± 2°C.
• 3.3. Winter temperature stimulated vitellogenin synthesis in midsummer, but winter photoperiod did not do so.
• 4.4. Captivity without food for 22 days reduced the rate of vitellogenin synthesis in summer but had no effect in winter.

     

Immunocytochemical localisation of vitellogenic and non-vitellogenic yolk proteins in the ovary of leptinotarsa decemlineata

• 1.1. Two vitellogenins and chromoprotein 2 are selectively accumulated by the oocyte and cannot be detected either in follicle cells or in the germarium.
• 2.2. At the start of their accumulation in terminal oocytes they are asymmetrically distributed.
• 3.3. Endocytosis of vitellogenin 1 starts somewhat later than the uptake of vitellogenin 2 and chromoprotein 2.
• 4.4. In follicle cells of young follicles, a protein (DLP), immunologically related to diapause protein 1, is highly concentrated.
• 5.5. During vitellogenesis DLP is sequestered by the oocytes.
• 6.6. The protein rich globules in terminal oocytes contain the vitellins as well as chromoprotein 2 and DLP.

     

Poly(d(A—T)) dependent RNA polymerase activity after treatment of nuclei with micrococcus nuclease

• 1.1. Rat liver nuclei were incubated with or without 20 units micrococcus nuclease (EC3.1.4.7)/mg nuclear DNA.
• 2.2. The soluble poly(d(A—T)) dependent RNA polymerases were reduced in activity to 15–20% that of the controls after treatment with micrococcus nuclease.
• 3.3. RNA polymerases I plus III activities were completely, RNA polymerase II activity partially reversible on removal of the DNA released into the soluble fraction by treatment of nuclei with micrococcus nuclease.
• 4.4. Inhibitory constants obtained with the solubilized DNA were 17.1 μM and 20.7 μM nucleotide-DNA for RNA polymerases I plus III and RNA polymerase II, respectively. The corresponding inhibitory constants obtained with native salmon DNA were 23.0 μM and 34.4 μM nucleotide-DNA.

     

Seasonal changes of total lipids in the turtle Sternotherus odoratus

• 1.1. Seasonal variation in total lipids was examined in several body components of the turtle Sternotherus odoratus.
• 2.2. Carcass fat stores in both sexes were depleted during winter. Additionally, a decline in carcass lipids was associated with increases in gonadal mass.
• 3.3. Concentrations of liver lipids were maximal during August and minimal during winter.
• 4.4. Males showed little seasonal change in plasma lipid levels, whereas females had seasonal peaks temporally associated with ovarian development and carcass fat storage.
• 5.5. Ovarian concentrations of lipids were minimal after nesting and increased during fall.
• 6.6. Results suggest that S. odoratus uses stored fats both for reproduction and maintenance during winter.

     

An electrophoretic comparison of non-histone proteins from rat liver total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins

• 1.1. A procedure of isolation of non-histone proteins from rat liver chromatin in mild conditions provided 3 groups of these proteins, i.e. NHCP1, NHCP2 and NHCP3.
• 2.2. The investigated proteins are devoid of DNA and revealed various influences on RNA synthesis in vitro.
• 3.3. The extraction of rat liver chromatin with 0.35 M NaCl (pH 7.5) removed about 30% of examined proteins. Electrophoretic patterns of 3 groups of non-histone proteins from total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins are compared.

     

Nucleic acids accumulation of silk gland of Bombyx mori in relation to silk protein

• 1.1. Productivity of silk, properly fibroin, of the silkworm Bombyx mori was in proportion to the amount of RNA accumulation in the posterior division of silk gland.
• 2.2. DNA content of the silk gland of a line of high silk productivity was twice as much as that of low productivity. A DNA molecule can transcribe RNA, ranged from 3 × 10⁶ to 6 × 10⁶ molecules.
• 3.3. An application of actinomycin to larvae lowered an accumulation of RNA in the silk gland and resulted in a decrease of silk production.
• 4.4. Even under the upper limit of starvation by which the larvae complete their life, the silk gland kept a normal level of the DNA content, except that it lost the synthesizing activity of RNA.
• 5.5. Treatment of larvae with juvenile hormone occasionally induced another DNA replication of the silk gland.

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

Proteome-wide Tyrosine Phosphorylation Analysis Reveals Dysregulated Signaling Pathways in Ovarian Tumors

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Highlights

• •Multiplexed PTM assays on HuProt array were developed using ovarian tumor lysates.
• •Proteome-wide Tyr phosphorylation with 102 ovarian tumors were performed and analyzed.
• •19 kinases were predicted to have elevated activities in ovarian tumor.
• •Elevated activities of PTK2 and PTK2B were confirmed in ovarian cancer cell lines.

     

Sex-specific androgen binding in spotted hyaena (Crocuta crocuta) plasma

• 1.1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas.
• 2.2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females.
• 3.3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17β binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species.
• 4.4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation.
• 5.5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.

     

Synthesis and maturation of the major yolk protein by the ovaries of the firebrat,Thermobia domestica (Insecta,thysanura)

• 1.1. Ovaries of Therobia domestica, dissected from inseminated females and incubated with tritiated amino acids, synthesize labeled proteins, the major fraction of which is indistinguishable from the major vitellogenin secreted by the fat body, when considering the electrophoretic mobility, the polypeptide composition and the immunoreactivity.
• 2.2. Peptide mapping, using two different proteases, shows a striking structural similarity between the proteins of both origins and reveals interrelationships between their subunits.
• 3.3. The ovary synthesizes the 210–212 kD precursors of the major vitellogenin, as does the fat body, and processes them intensively into smaller subunits (176–182, 57 and 46 kD). The follicle cells are tentatively nominated for both roles.
• 4.4. The quantitative contribution of the two ovaries to the vitellogenin pool was found to be much higher than that of the fat body.