High molecular weight messenger rna in polysomes of osmotic dependent saccharomyces cerevisiae mutants

• 1.1. A fraction of heavy polysomes has been isolated from rapidly dividing osmotic dependent yeast cells. Poly A + RNA purified from this fraction represent 30% of the total polysomal poly A + RNA and consist of high molecular weight molecules.
• 2.2. Both centrifugation analysis in denaturing conditions and electron microscopic studies showed the existence of mRNA with molecular weight up to 5 × 10⁶ dallons in polysomal fractions.
• 3.3. Competition hybridization experiments suggested a considerable sequence homology between high and low molecular weight poly A + RNA purified from heavy and light polysomal fractions, respectively.
• 4.4. These results suggest that in rapid growing yeast cells some of the abundant mRNA might be synthesized by a mode different from the monocistronic mRNA synthesis.

     

Isolation and partial characterization of vitellin from the egg of the giant tiger prawn,penaeus monodon

• 1.1. The results obtained from the present study show that vitellin (yolk protein), a glycolipoprotein derived from the giant tiger prawn, Penaeus monodon, consists of four polypeptides, Ep1, Ep2, Ep3 and Ep4, with molecular weights of 168, 104, 83 and 74 kDa, respectively. This protein was purified using SDS-PAGE and gel-elution.
• 2.2. Antisera against Ep2, Ep3 and Ep4 fractions showed very strong specific binding reactivity when reacted with shrimp ovaries at developing stages.
• 3.3. No crossreaction was observed among Ep2, Ep3 and Ep4 fractions of vitellin. However, anti-Ep2 and anti-Ep3 were demonstrated to be able to react with Ep1 fraction. The homogenates obtained from ripe ovaries and hemolymph of vitllogenesis females showed an immuno-identical pattern to those obtained from egg extracts.
• 4.4. The proteolysis mapping for each fraction showed that Ep1, the high molecular weight fraction, contained Ep2 and Ep3 fragments. These results suggest that Ep1 may be a precursor for Ep2 and Ep3.

     

The stimulation of yeast mitochondrial protein synthesis by low molecular weight cytoplasmic factors: Requirement for intact mitochondria and lack of effect by folate derivatives

• 1.1. Protein synthesis by GTP -supplemented yeast mitochondria is stimulated by a fraction of molecular weight less than 2,000 isolated from yeast high-speed supernatant (S-150).
• 2.2. The low molecular weight fraction works independently of the respiratory chain as the stimulation effect is not cyanide-sensitive.
• 3.3. Stimulation of mitochondrial protein synthesis by cytoplasmic factors is dependent upon the method of mitochondrial isolation.
• 4.4. The low molecular weight stimulatory factor(s) are not reduced folate derivatives which supply formyl groups required for initiation of mitochondrial protein synthesis.

     

Subcellular distribution of metals within the kidney of the bivalve Mercenaria mercenaria (L.)

• 1.1. The subcellular distribution of nine transition metals (plus four additional elements) was measured in the kidney tissue of the quahog, Mercenaria mercenaria.
• 2.2. Elemental analyses of the subcellular fractions indicated three main patterns of metal distribution within kidney cells.
• 3.3. Barium, iron, manganese and lead were associated primarily with kidney granules.
• 4.4. Cadmium, copper, potassium and magnesium were found mainly in the cytosolic fraction.
• 5.5. Calcium, phosphorus and zinc were found in all isolated fractions, probably reflecting the important roles that these elements play in bivalve metabolism.
• 6.6. The organelle composition of the isolated subcellular fractions was determined using marker enzyme assays and microscopic techniques.

     

Protein differences in cilia of mechanoreceptor hair and gill cells of the scallop Mizuhopecten yessoensis

• 1.1. In search for mechanosensory molecules the composition of the ciliary proteins of mechanoreceptor hair cells of the abdominal organ and less mechanosensitive gill cells were compared electrophoretically.
• 2.2. The hair cells and gill cilia were very similar in their polypeptide sets but differed by contents of three axonemal polypeptides with molecular weights of 125, 149 and 300 kilodaltons (kDa) and one membrane polypeptide of 159 kDa.
• 3.3. The membrane polypeptide with a molecular weight of 159 kDa represented approximately 3% of the total ciliary protein of hair cells. There was only a trace of this polypeptide in gill cilia and their membrane fraction.
• 4.4. A peculiarity of ciliary membranes of the hair cells was a high content of the 159 kDa-polypeptide, which constituted more than 20% of the total protein of membrane fraction.

     

Transport of cholesterol in the hemolymph of the mollusc Diplodon delodontus

• 1.1. The dietary and inter-organ cholesterol transport in the hemolymph of the bivalve mollusc Diplodon delodontus, was studied. Plasma and hemocytes were obtained after feeding labeled cholesterol to animals or injecting it into the posterior adductor muscle.
• 2.2. In both cases, cholesterol was incorporated either into plasma or hematic cells.
• 3.3. Two plasmatic fractions differing in their hydrated densities were recognized as cholesterol carriers and were isolated. They have characteristics of high density (HDL) and very high density (VHDL) lipoproteins, respectively.
• 4.4. The major lipids in the different classes of lipoproteins were free sterols in HDL and phospholipids in VHDL.
• 5.5. Neither low nor very low density lipoprotein transporting cholesterol was detected.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Biochemical variations in three species of prairie dogs (Cynomys)

• 1.1. Blood proteins were studied by polyacrylamide disc gel electrophoresis in three species of prairie dogs, Cynomys gunnisoni, C. leucurus, and C. ludovicianus.
• 2.2. The sera were separated into 13–15 fractions and the three species could be distinguished by both qualitative and quantitative differences in their serum patterns.
• 3.3. Qualitatively, variations in the occurrence and number of slow albumin fractions are diagnostic at the species leel.
• 4.4. Quantitative differences were most apparent in variation in the mobility of the major albumin fraction and the transferrin fraction.

     

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

• 1.1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments.
• 2.2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long.
• 3.3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction.
• 4.4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Lipoxygenase of Thermoactinomyces vulgaris,purification and characterization of reaction products

• 1.1. A lipoxygenase preparation was obtained from Thermoactinomyces vulgaris and was purified by affinity chromatography on a linoleyl aminoethyl sepharose column.
• 2.2. Two active fractions were obtained.
• 3.3. The fraction obtained by elution with 100 mM borate buffer pH 9.0 was used in the subsequent work.
• 4.4. Th. vulgaris lipoxygenase oxidized linoleic acid into two products: 13-HPOD and 9-HPOD at a ratio of 44 to 56, respectively.
• 5.5. The identification and characterization of the isomers was done by HPLC, I.R. and mass spectrometry.
• 6.6. When arachidonic acid was used as substrate, 15-HPETE and 15-HETE were found to be the main enzymatic products.

     

Structural features of carbohydrate chains in human salivary mucins

• 1.1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated.
• 2.2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units.
• 3.3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants.
• 4.4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine.
• 5.5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

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.

     

Lipid agglutinins from coelomic fluid of the earthworm,Lumbricus terrestris

• 1.1. Coelomic fluid of the earthworm, Lumbricus terrestris, contains lipid agglutinins that are primarily glycolipids. They are present in fluid from both immunized (induced) and unimmunized (naturally occurring) worms.
• 2.2. Lumbricus agglutinins partially purified by Folch extraction followed by silicic acid chromatography, were present in all fractions but in highest concentration in acetone and methanol fractions.
• 3.3. Immunodetection revealed agglutinin activity in acetone and methanol fractions, but not in the chloroform fraction.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Morphometry and composition of red drum otoliths: Changes associated with temperature,somatic growth rate,and age

• 1.1. Size and composition of sagittal otoliths from red drum, Sciaenops ocellatus (Sciaenidae), reared at various constant temperatures were compared with otoliths from wild-caught fish.
• 2.2. Uncoupling of otolith growth and somatic growth in laboratory-reared fish was evident in otolith length, area, volume, weight, density, and organic fraction.
• 3.3. Fish grown at low temperatures had significantly smaller and less dense otoliths having a greater organic content than fish of the same size grown at higher temperatures.
• 4.4. Changes in inorganic elements were poorly related to temperature in laboratory-reared fish.
• 5.5. The effect of temperature on otolith elemental composition was small relative to the effects of age and its associated physiological changes.

     

Purification and partial characterization of 200 kDa oncofetal antigen from radiation induced murine lymphocytic lymphoma

• 1.1. A 200 kDa glycoprotein (gp200) oncofetal antigen was purified from solubilized membranes of a radiation-induced murine lymphomcytic lymphoma cell line (XR11-5T), grown in syngeneic RFM mice, by successive gel chromatography of the active fraction on lentil lectin agarose, Q- and S-Sepharose and Superose-12 using an FPLC system.
• 2.2. A murine monoclonal antibody 115, produced by the syngeneic immunization of adult male C57BL/6N mice with 12-day mouse fetal cells, was used in a slot blot antibody assay to follow up the active fractions.
• 3.3. The purified glycoprotein has a pi of 5.4.
• 4.4. Treatment of radiolabeled gp200 with neuraminidase caused a slight reduction in size due to the removal of sialic acid groups and a shift in pI to 6.3.
• 5.5. Treatment of gp200 with different glycosidases shows that gp200 is susceptible to N- and O-glycanase but not to endoglycosaminidase H.
• 6.6. On extraction of gp200 with Triton X-114 it partitions exclusively into the detergent-rich fraction consistent with being an integral membrane protein.