Small RNAs in Bombyx mori nuclear polyhedrosis virus polyhedra form complexes with polypeptide p14 and polyhedrin

It was shown that two small RNAs about 65 and 55 nucleotides long included in NPV B. mori polyhedra form with polypeptides p29 and p14 specific RNP-complexes with molecular weights of 50 and 31 kDa, respectively. Both complexes form high-molecular weight complex with polyhedrin. Origin and nature of p29 and p14 polypeptides are discussed.     

Purification of a high molecular weight somatomedin binding protein from human plasma

The somatomedins insulin-like growth factor I and II (1,2) are in serum bound to high-molecular weight binding proteins (6,7,8). By use of a four step chromatographic procedure a somatomedin binding protein was isolated from outdated human plasma. Exclusion chromatography on Sephadex G-200 disclosed a molecular weight of 150 kDa. After lyophilization however, the binding activity was found in a lower molecular weight range of 35-45 kDa. A partial amino acid sequence analysis of the lyophilized material revealed a possible N-terminal sequence of Ala-Pro-Trp. This sequence is identical to the N-terminal sequence of the 35 kDa somatomedin binding protein previously isolated from human amniotic fluid (16).     

RNP-complex stoichiometry of Bombyx mori nuclear polyhedrosis virus polyhedra

By gel-filtration through Sephacryl S-300 it was shown that RNP A complex present in polyhedra of Bombyx mori nuclear polyhedrosis virus has molecular weight (M(w)) about 700 kDa. It was shown that RNP A with M(w) 788 kDa is composed of two polyhedrin 13S-associates with M(w) 342 kDa, two p14 polypeptide with M(w) 14 kDa, two 21 kDa small non-coded RNAs and two 17 kDa small non-coded RNAs. The model of RNP A formation from components making it is proposed. The complex role in the course of polyhedron formation and its role in the course of infection are discussed.     

Isoforms of trehalase and invertase of Fusarium oxysporum

Enzymatic assays and native PAGE were used to study trehalase and invertase activities, depending on culture age and different sugar conditions, in cell-free extracts, culture filtrates and ribosomal wash of Fusarium oxysporum. The activity of invertase preceded that of trehalase; in the exponential phase of growth, mainly invertase activity was produced, whereas trehalase activity was high in the stationary phase. In this last phase of growth, the activity of intracellular trehalase was repressed by monosaccharides, whereas disaccharides, especially lactose and starch, enhanced the activity of intracellular and extracellular trehalase. However, invertase activity was not repressed under these conditions and had the maximal activity in the presence of saccharose. Intracellular trehalase appeared in a single, high-molecular weight (120 kDa) form, whereas the extracellular enzyme appeared in a single, low-molecular weight (60 kDa) form. The activity pattern of invertase isoforms indicated the occurrence of three forms of intracellular enzyme with the main activity band at 120 kDa and two isoforms of extracellular enzyme. In the ribosomal wash, high-molecular weight isoforms of both trehalase and invertase were identified. A possible role of trehalase and invertase in carbohydrate metabolism of fungal pathogens is also discussed.     

Effect of Molecular Weight and Degree of Acetylation on Adjuvantive Properties of Chitosan Derivatives

The hemostatic and immunostimulating activity and cytotoxicity were determined for a number of chitosans differing in molecular weight (from 3 to 510 kDa) and degree of acetylation (from 1 to 25 mol%) that were used as adjuvants in inactivated poliomyelitic, influenza, and live influenza vaccines. It has been shown that the hemostatic activity of chitosan increased sharply with an increase in its molecular weight. In oligochitosan with a molecular weight of <16 kDa, it was smaller by a factor of 15–100 than in chitosan with a molecular weight of 20–510 kDa. The level of increase in the immunogenicity of vaccines containing oligochitosan as adjuvants was not lower than that for the vaccine including high-molecular chitosan. However, the immunostimulatory activity of oligochitosan depended on the degree of acetylation, reaching a maximum value at 6 mol%. It was shown that all oligochitosans and chitosans with a molecular mass below ~50 kDa showed almost no cytotoxicity at a concentration of ≤2.5 mg/mL, which enable their use as adjuvants for inactivated and live vaccines at the optimal ratio of molecular weight to the degree of acetylation.     

Modified arabinogalactans from callus culture <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G.

The cultivation of Silene vulgaris (M.) G. callus culture on the nutrient mediums contained carbohydrates, phytohormones, nitrogen, and phosphate has led to the modification of the arabinogalactan structure from the cell walls. It was noticed that a sucrose concentration increase in the cultivation medium led to an increase of the arabinogalactan fragment yield with a molecular weight more than 300 kDa and a decrease of the yield of fragments with molecular weight less than 300 kDa. The sucrose concentration increase in the nutrient medium entailed the increase of arabinose and galactose content in the fragment with the molecular weight more than 300 kDa and a decrease in the fragment with a molecular weight of 100–300 kDa. On the nutrient medium containing a mix of sucrose and arabinose, the yield of the fraction with a molecular weight more than 300 kDa and the amount of arabinose residues increased, and the yield of minor fragments and the content of arabinose and galactose residues, included in these, decreased. On the medium containing an increased concentration of 2,4-dichlorphenoxyacetic acid, the yield of high-molecular fragment and the content of arabinose residues are two times increased. The decreasing of the amount of arabinose and galactose residues in the fragment with a molecular weight more than 300 kDa was observed at a lack of nitrogen or phosphate in the nutrient medium.     

Transformation of humic acids by two-domain laccase from Streptomyces anulatus

The properties of two-domain laccase of Streptomyces anulatus (SaSL) and its role in transformation of humic acids (HA) from chernozem, sod-podzolic soil and peat at alkaline pH values were studied. The SaSL was cloned, expressed in E. coli and obtained in an electrophoretically homogeneous state. SaSL is an oligomeric protein with a molecular weight of 235-300 kDa and six or eight subunits in the molecule. The molecular weight of the subunit is 37.7 kDa. Its catalytic properties are similar to those of the previously described two-domain laccase. The enzyme catalyzed oxidation of electron donors (K₄[Fe(CN)₆], ABTS) at acidic pH and phenolic substrates (2-methoxyphenol, 2,6-dimethixyphenol) at alkaline pH values. The efficiency of catalysis was higher in the case of electron donors than phenolic substrates. The enzyme showed a high thermal stability and was more stable at neutral and alkaline pH values. The enzyme effectively transformed humic acids at alkaline pH values. It was found that polymerization reactions took place during interaction of SaSL with HA, as well as with their high-molecular weight (>80 kDa) and low-molecular weight (<5 kDa) fractions. Our results suggest a possible involvement of the two-domaim laccases in humification processes in alkaline soils.     

Acid phosphatases in mammalian tissues. Evidence for the existence of a 57 kDa Zn(2+)-dependent acid phosphatase form.

1. A comparative study of multiple forms of acid phosphatase (AcPase) in various organs of mammals was carried out. 2. These studies indicated that the high-molecular weight AcPase is preferentially expressed by tissues which undergo cell proliferation such as epithelial tissues; on the contrary, the low-molecular weight enzyme seems to be characteristic of highly differentiated tissues such as nervous, muscle and blood erythrocytes. 3. The existence of a new AcPase activated by Zn2+ ions was observed in all tissues studied with the exception of erythrocytes. 4. The enzyme shows a molecular weight of 57 kDa, is insensitive to NaF, hydrolyzes p-nitro-phenylphosphate and o-c-phenylphosphate; ATP, a-naphthyl-phosphate and beta-glycerolphosphate are also dephosphorylated.     

The chemical nature of the fluorescing products accumulating in the lipids of the crystalline lenses of mice with hereditary cataract

The content of diene conjugates (lipid hydroperoxides) was shown to be significantly higher in lipids extracted from the lenses of mice with hereditary cataract than in the controls. The same holds true for characteristics of fluorescence of the end-product of lipid peroxidation. Two (low- and high-molecular weight) peaks were detected in chromatographic lipid profile of cataract lenses measured by fluorescence on Sephadex LH-20 column, whereas only one (high-molecular weight) peak was found in lipids from normal lenses. It was established that high-molecular weight fluorescent fractions corresponded to lipid components of lipofuscin-like pigments. NMR and mass spectrometry of low-molecular weight fractions suggested that they contained predominantly products of free radical oxidation of long chain polyunsaturated fatty acids (C22:6).     

Semitelechelic HPMA copolymers functionalized with triphenylphosphonium as drug carriers for membrane transduction and mitochondrial localization

Semitelechelic HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers possessing a single terminal lipophilic triphenylphosphonium (TPP) cation and fluorescent labels were synthesized to determine how the attached cation affected cellular uptake and intracellular trafficking. In vitro mitochondrial uptake fluorescence quenching assays using isolated mouse liver mitochondria indicated that only lower molecular weight (<5 kDa) BODIPY FL-labeled TPP-semitelechelic HPMA copolymers exhibited significant organelle localization or uptake. In vitro cellular uptake and intracellular trafficking was evaluated using cultured human ovarian carcinoma cells. Cells incubated with all types of TPP copolymers used in the study appeared to internalize the polymer by endocytosis only, and all of the internalized copolymer was confined to the lysosomal compartment after 24 h. Endocytotic uptake of the TPP-HPMA copolymer conjugates was rapid, suggesting that they were internalized by adsorptive endocytosis, rather than fluid-phase pinocytosis. Low-molecular weight (<5 kDa) and high-molecular weight (>5 kDa) semitelechelic copolymers, microinjected into cultured cells indicated that the TPP moiety did not significantly localize the polymers to mitochondria.     

The activity of postural asymmetry factors in symmetrical sections of the rat spinal cord

The distribution of the low-molecular weight and high-molecular weight postural asymmetry factors (FPA) activity in the left and right parts of the lumbal region of the rat spinal cord was studied. Low-molecular weight FPA induces flexion of the hind limb ipsilateral to the half of the spinal cord from which FPA was isolated, while high-molecular weight FPA induces contralateral flexion. The activities of the low- and high-molecular weight FPAs in each half of the spinal cord are comparable in normal rat. After the suction lesion of the motor areas in the left hemisphere the increase of the low-molecular weight FPA activity in the right half of the lumbal region of the spinal cord was observed.     

Purification of cytochrome B-561 from chromaffin granules and its physico-chemical properties

A soft method of purification of cytochrome-561 from the membranes of chromaffin granules has been developed. It permits isolating a protein in its natural microsurroundings, i.e. a complex with lipids, provided that a buffer with high ionic force is used without a detergent. This method helps obtaining an electrophoretically homogeneous preparation as a high-molecular lipoprotein hexamer whose molecular weight is about 400 kDa. Basic physicochemical parameters of this preparation (subunit composition, content and composition of lipids, heme content, spectra of optical absorption of the oxidized and reduced forms) are determined. Possible presence of two forms of cytochrome b-561 in the chromaffin granules is discussed.     

Altered chaperone and protein turnover regulators expression in cultured skin fibroblasts from type 1 diabetes mellitus with nephropathy

In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM.     

Immunohistochemical localization of a tenascin-like extracellular matrix protein in sea urchin embryos

Summary We have used polyclonal antisera raised against vertebrate tenascin to identify and localize tenascin-like proteins in the developing sea urchin. These antisera recognize high-molecular weight proteins on immunoblots of sea urchin embryo homogenates that are similar in size and appearance to tenascin from vertebrates. These proteins appear as a doublet with an apparent molecular weight of 150 kDa and a larger, broad band with an apparent molecular weight of 350 kDa. Whole mounts of sea urchin embryos and larvae were stained with one of these antisera. The anti-tenascin stained the surface of primary mesenchyme cells during their phase of active migration. This staining was sensitive to detergent, suggesting that the protein recognized by the antiserum was associated with the cell surface. During later stages of development, the bulk of the antitenascin staining was found dispersed throughout the blastocoel matrix, and was no longer sensitive to detergent. We conclude that sea urchins express tenascin-like proteins during early stages of development, and that these proteins may play a role associated with primary mesenchyme cell morphogenesis.     

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg² was required foractivity, the activity with Mn², Co² and Zn² was 48, 48 and 182% of the activity observed with Mg², respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent K_m for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.     

Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).     

A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.     

Glycosylation of erythrocyte spectrin and its modification in visceral leishmaniasis

Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBC(VL)). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and β-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrin(VL)) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and β-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrin(N)) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrin(N). Although the presence of both N- and O-glycosylations was found both in spectrin(N) and spectrin(VL), enhanced sialylation was predominantly induced in spectrin(VL). Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and β-spectrin(VL) confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrin(VL) showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrin(N) suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBC(VL). The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrin(VL) as evidenced by the presence of an additional 60 kDa fragment, absent in spectrin(N) which possibly affects the biology of RBC(VL) linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.