DNA polymerases of Euglena gracilis: heterogeneity of molecular weight and subunit structure.

Sedimentation analysis of glycerol-density gradients has shown that freshly purified DNA polymerases A and B (pol A and pol B) of Euglena gracilis have molecular weights of 185,000 (8.7S) and 240,000 (10.3S) respectively. They can aggregate in fresh preparations to give forms of higher molecular weight as shown by gel filtration through Sepharose 6B, but on ageing pol B progressively generates species with sedimentation coefficients of 7.4-7.7S, 6.3-6.5S, 4.8S and finally 3.0S. Pol A apparently behaves in a similar fashion though it is unstable. Exposure of pol A and pol B to high ionic strengths can also cause their breakdown to species with lower sedimentation coefficients. The mitochondrial DNA polymerase is distinct, having a molecular weight of 170,000. It is proposed that pol A and pol B are oligomers of the 3.0S subunit and possibly other dissimilar subunits, with pol B having additional factors conferring upon it its extra catalytic functions.     

Hyaluronic acid: molecular conformations and interactions in two sodium salts.

A detailed structure for the tetragonal form (a = b = 0.989 nm, c, fibre axis, = 3.394 nm) of sodium hyaluronate has been obtained by analysing X-ray fibre diffraction data using new molecular modelling techniques. Two polysaccharide chains pass through each unit cell, one at the corner and one at the centre. The chains are anti-parallel to one another. Each chain is a left-handed, 4-fold helix of disaccharide units. There are intramolecular hydrogen bonds stabilising each glycosidic linkage. Octahedrally co-ordinated sodium ions link, by O … Na⁺ … O bridges, neighbouring polysaccharide chains that are further linked by hydrogen bonds. No double-helix model (as originally proposed for this structure) has been found to be free of unacceptable non-bonded contacts or to fit the diffraction intensities as closely.The tetragonal form, which is stable at zero relative humidity, contains no detectable water molecules. At higher relative humidities a related orthorhombic form is observed in which only the a dimension of the lattice is different (a = 1.153 nm, b = 0.989 nm, c = 3.386 nm). In this form the hyaluronate helix is 2-fold with tetrasaccharide units conformationally similar to the 4-fold helix of the tetragonal form. The Na⁺ … O binding and hydrogen bonds lost on expansion of the tetragonal lattice are all replaced in the orthorhombic structure by bridges through water molecules, four of which associated with each tetrasaccharide.     

Hyaluronic acid N-deacetylase assay in whole skin

Hyaluronic acid (HA) N-deacetylase(s) was quantified in whole skin, using a novel method that involved reaction of skin with exogenous HA as substrate. Acetyl (CH(3)CO-) moieties generated were converted chemically to MeOAc and quantified using gas chromatography/mass spectrometry. HA (1.7 mg) and skin (1.0 g) yielded 3.32 and 769.00 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively. Without added HA, 194.00 microg of product was obtained from the 76.5-year-old-patient samples. With chondroitin as substrate, the yields were 2.89 and 818.04 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively. The K5 (capsular, Escherichia coli polysaccharide) substrate yielded no detectable product, except for 170.02 microg from the 76.5-year-old-patient samples. This highly sensitive method was used to demonstrate that human-skin-HA N-deacetylase(s) was first detectable at 69 years of age, highly active at 76.5 years of age, and specific for N-acetyl moieties of d-GlcNAc and d-GalNAc where C(1) is beta-linked as in HA and CH.     

Hyaluronic acid of high molecular weight inhibits proliferation and induces cell death in U937 macrophage cells

Hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix, has regulatory influences on cells and cellular activities. To explore the effects of a high concentration (1 mg/mL) of high molecular weight HA (500-730 kD) on U937 macrophage growth dynamics, three factors that influence overall cellular growth, namely proliferation, apoptosis, and cell death, were examined. Cells were cultured with HA and were analyzed by flow cytometry every 24 hours during a 168-hour period for proliferation and the presence of apoptotic and dead cells. These analyses demonstrated that HA inhibits U937 macrophage proliferation in a time-dependent manner. Through the first 72 hours, cells exhibited slowed proliferation. However, no evidence of cell division arrest or reduced cell viability was observed. Thereafter, HA continued to diminish proliferation, but induced apoptosis. This data is consistent with regulatory influences secondary to HA binding to CD44 and/or RHAMM cell surface receptors, both of which were shown to be expressed on U937 macrophages. This study demonstrates that a high concentration of high molecular weight HA greatly inhibits macrophage population growth by the dual actions of impeding cell proliferation and inducing apoptosis.     

Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynthesis pathway

Hyaluronic acid (HA) production in Streptococcus zooepidemicus competes for the carbon source along with biomass formation, lactate formation (via glycolysis) and pentose phosphate pathway (PPP). In our studies, increase in HA molecular weight was observed by redirecting the carbon flux towards HA biosynthesis pathway by partially inhibiting the glycolytic pathway. Batch bioreactor (1.2 L) studies showed that with the addition of 25 μM sodium iodoacetate, 5 g/L tryptophan and 10 g/L pyruvate, which are glycolytic inhibitors, HA molecular weight increased to 3.2, 3.2 and 3.1 MDa respectively compared to control run (2.4 MDa). Yield coefficients Y_HA/S and Y_LA/S showed inverse relationship, indicating competition for glucose between HA and lactic acid formation. Addition of 5 g/L glutamine along with 25 μM sodium iodoacetate also increased the HA concentration to 5.0 g/L from 2.0 g/L in control run. Metabolic flux analysis studies show that concentration and molecular weight of HA is increased by decreasing carbon flux towards glycolysis and PPP and increasing carbon flux towards HA precursor formation. It was observed that specific growth rate of the cells correlated positively to the specific HA production rate and negatively to the molecular weight of HA produced. Addition of antioxidant tannic acid also increased molecular weight to 3.0 MDa.     

Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).     

Purification and characterization of iduronic acid-rich and glucuronic acid-rich proteoglycans implicated in human post-burn keloid scar

Small proteoglycans (PGs), extracted from human keloid scar tissue with 4M guanidinium chloride and fractionated by DEAE-cellulose chromatography, were separated by ethanol precipitation into one L-iduronic acid-rich and one D-glucuronic acid-rich fraction. The size of the L-iduronic acid-rich PG was 102 kDa with a 27 kDa glycosaminoglycan chain, that of the D-glucuronic acid-rich PG was 90 kDa with a 26 kDa glycosaminoglycan chain, and the protein core of both PGs was 14.5 kDa. The two PGs carried sulfate groups mostly attached at C-4 of the 2-amino-2-deoxy-D-galactose units. The N-terminal amino acid sequence of both was similar to human bone PGII (decorin), normal and hypertrophic scar, and human dermal tissue PG.     

Expression and localization of insulin-like growth factor-1 in normal and post-burn hypertrophic scar tissue in human

The migration of epithelial cells from dermal appendages toward the wound surface is essential for re-epithelialization of partial thickness burn injuries. This study provides evidence that these cells in vivo synthesize a mitogenic and fibrogenic factor, insulin-like growth factor-1 (IGF-1), which may promote the development of the post-burn fibroproliferative disorder, hypertrophic scarring (HSc). An evaluation of 7 post-burn hypertrophic scars, 7 normal skin samples obtained from the same patients and 4 mature scars revealed that IGF-1 expressing cells from the disrupted sweat glands tend to reform small sweat glands of 4-10 cells/gland in post-burn HSc. The number of these cells increases with time and the glands become larger in mature scar. Other epithelial cells such as those found in sebaceous glands and basal and suprabasal keratinocytes, also express IGF-1 protein and mRNA as detected by Northern and RT-PCR analysis of RNA obtained from whole skin and separated epidermis and dermis. However, cultured keratinocytes did not express mRNA for IGF-1. Histological comparisons between normal and HSc sections show no mature sebaceous glands in dermal fibrotic tissues but the number of IGF-1 producing cells including infiltrated immune cells was markedly higher in the dermis of hypertrophic scar tissues relative to that of the normal control. In these tissues, but not in normal dermis, IGF-1 protein was found associated with the extracellular matrix. By in situ hybridization, IGF-1 mRNA was localized to both epithelial and infiltrated immune cells. Collectively, these findings suggest that in normal skin, fibroblasts have little or no access to diffusible IGF-1 expressed by epithelial cells of the epidermis, sweat and sebaceous glands; while following dermal injury when these structures are disrupted, IGF-1 may contribute to the development of fibrosis through its fibrogenic and mitogenic functions. Reformation of sweat glands during the later stages of healing may, therefore, limit this accessibility, and lead to scar maturation.     

Alpha 1-antitrypsin: apparent molecular weight heterogeneity shown by two-dimensional electrophoresis.

Two-dimensional (2-D) gel electrophoresis was used to examine charge and molecular weight variability of alpha 1-antitrypsin. Two-D electrophoresis resolved distinctive differences among individual phenotypes. Microheterogeneity of charge was seen for the different alleles that corresponded to the charge variability observed on isoelectric focusing gels. The molecular weights of the major components of each allele appeared to differ from each other by approximately 1,000, suggesting, that in addition to sialic acid, there may be differences in neutral sugar composition between the individual components. In comparison to the M allele components, the corresponding S and Z components had higher molecular weights. The MZ and MS phenotypes showed characteristic patterns of protein spot doublets. Computerized quantitation was used to separate and estimate the contribution of each component to the overall allele composition. The Z allele components contained about 15% of the total MZ quantity. The 2-D electrophoresis technique may offer a new approach for molecular structural studies of alpha 1-antitrypsin variants and similar glycoproteins.     

Determination of molecular weight and molecular structure of rat-liver pyruvate carboxylase.

The molecular weight of pyruvate carboxylase isolated from pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and electron microscopy. The enzyme molecule appeared as a tetramer with the four subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a molecular weight Mc=0r of 280 000 and an So20,w of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4 degrees C or incubation at -- 20 degrees C at low protein concentrations. The isolated trimers and dimers had a molecular weight Mc=0r of 210 000 and 140 000, respectively, and an So20,w of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20 degrees C yielded enzymically inactive subunits (Mc=0r = 70 000; So20,w = 4.95 S). The molecular weights (for pyruvate carboxylase and its subunits), as calculated from the subunit diameter observed in the electron microscope, were consistent with the values obtained from sedimentation studies.     

Hyaluronic acid in human articular cartilage. Age-related changes in content and size.

A specific lactotransferrin receptor was identified in the mouse small-intestinal brush-border membrane and the binding features were investigated in homologous and heterologous systems. The receptor was found to be specific for lactotransferrins isolated from milk of various species, but the affinity was higher toward the homologous ligand (Ka = 3.5 x 10(6) M-1 compared with 2.6 x 10(6) M-1 for both human and bovine lactotransferrins). However, the number of binding sites (n) was the same for the three lactotransferrins, namely 0.53 x 10(12)/micrograms of membrane protein. The binding of mouse lactotransferrin to its receptor was found to be pH-dependent, with an optimal binding at pH 5.5, and seemed unlikely to be carbohydrate-mediated. The receptor was demonstrated to be devoid of any affinity for human and mouse serotransferrins or for a 'serotransferrin-like' protein isolated from mouse milk. The receptor was solubilized with 1% Triton X-100 with good yield. The solubilized receptor was found to retain lactotransferrin-binding activity and sensitivity to pH.     

Interindividual heterogeneity of molecular weight of human brain neutral sphingomyelinase determined by radiation inactivation method

The molecular weight (M_r) of the membrane-bound neutral sphingomyelinase from human brain was determined using the radiation inactivation procedure. Previous studies on three human brains suggested a M_r of 165±25 kDa (J. Neurochem. 1985, 45: 630–632). We now report that in another human brain the neutral sphingomyelinase had a M_r of 740±100 kDa; this higher M_r was not accompanied by differences in enzymatic properties nor heat-stability.