Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications

Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-d-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.     

Augmented Insulinotropic Action of Arachidonic Acid through the Lipoxygenase Pathway in the Obese Zucker Rat

Objective: The metabolism of arachidonic acid (AA) has been shown to be altered in severe insulin resistance that is present in obese (fa/fa) Zucker rats. We examined the effects and mechanism of action of AA on basal and glucose‐stimulated insulin secretion in pancreatic islets isolated from obese (fa/fa) Zucker rats and their homozygous lean (Fa/Fa) littermates. Research Methods and Procedures: Islets were isolated from 10‐ to 12‐week‐old rats and incubated for 45 minutes in glucose concentrations ranging from 3.3 to 16.7 mM with or without inhibitors of the cyclooxygenase or lipoxygenase pathways. Medium insulin concentrations were measured by radioimmunoassay, and islet production of the 12‐lipoxygenase metabolite, 12‐hydroxyeicosatetraenoic acid (12‐HETE), was measured by enzyme immunoassay. Results: In islets from lean animals, AA stimulated insulin secretion at submaximally stimulatory glucose levels (< 11.1 mM) but not at 16.7 mM glucose. In contrast, in islets derived from obese rats, AA potentiated insulin secretion at all glucose concentrations. AA‐induced insulin secretion was augmented in islets from obese compared with lean rats at high concentrations of AA in the presence of 3.3 mM glucose. Furthermore, the inhibitor of 12‐lipoxygenase, esculetin (0.5 μM), inhibited AA‐stimulated insulin secretion in islets from obese but not lean rats. Finally, the islet production of the 12‐HETE was markedly enhanced in islets from obese rats, both in response to 16.7 mM glucose and to AA. Discussion: The insulin secretory response to AA is augmented in islets from obese Zucker rats by a mechanism related to enhanced activity of the 12‐lipoxygenase pathway. Therefore, augmented action of AA may be a mechanism underlying the adaptation of insulin secretion to the increased demand caused by insulin resistance in these animals.     

HIV-2 Integrase Variation in Integrase Inhibitor-Na?ve Adults in Senegal,West Africa

Background

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.

Methods

We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.

Results

No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.

Conclusion

Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.     

Class I-like MHC molecules expressed by baboon placental syncytiotrophoblast

Human placental villous trophoblast is known to be unreactive with W6/32 and other monoclonal antibodies recognizing monomorphic determinants of human class I MHC heavy chains, whereas extravillous cytotrophoblast in the placental bed is W6/32-reactive by immunohistology. We have now demonstrated, in contrast, that syncytiotrophoblast is the only cellular component of baboon early placental villous tissue which is reactive with any of these antibodies. Radioimmunoprecipitation of detergent-solubilized baboon placental membrane preparations, and subsequent SDS-PAGE, has shown the W6/32-reactive component to have an m.w. of 41,000 and to be associated with beta 2-microglobulin, whereas baboon peripheral lymphocytes express 45,000 m.w. W6/32-reactive antigens comparable with the HLA-A,B,C heavy chains of human lymphocytes.     

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25⁺ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

Gene by smoking interaction: evidence for effects on low-density lipoprotein size and plasma levels of triglyceride and high-density lipoprotein cholesterol

We seek to determine whether significant gene x smoking interaction effects exist on plasma triglyceride (TG) levels, HDL cholesterol (HDL-C) level, and median LDL particle diameter (LDL-MPD) in Mexican American families enrolled in the San Antonio Family Heart Study. The sample consisted of 1,392 individuals distributed in 42 extended pedigrees, ranging in age from 16 years to 92 years. Separate quantitative genetic analyses were carried out for TG and HDL-C level and LDL-MPD using a maximum-likelihood-based variance decomposition approach while simultaneously adjusting for age and sex. Initial heritability estimates demonstrated significant (p < 0.001) additive genetic contributions to all three traits (h2 range 0.50 - 0.54). To test for a gene x smoking interaction, we included in the model additional smoking-status-specific variance terms and a genetic correlation term between smokers and nonsmokers. Comparisons of nested models revealed significant evidence (p < 0.01) for a gene X smoking interaction effect on TG level and LDL-MPD and possible evidence for an effect on HDL-C level. These results indicate that the gene or suite of genes regulating each of these phenotypes is likely the same in smokers and non-smokers but that smoking may alter the expression of genes, particularly those influencing TG level and LDL-MPD.     

The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses.     

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5′ to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl₂ and CaCl₂. Although Mg²⁺- and Ca²⁺-activated CSP41 cleave at identical sites in the single-stranded regions of a stem–loop-containing substrate, Mg²⁺-activated CSP41 was also able to cleave within the double-stranded region of the stem–loop. Mixed metal experiments with Mg²⁺ and Ca²⁺ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn²⁺, Co²⁺ and Zn²⁺ compete with Mg²⁺ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg²⁺ (K_A,Mg = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg²⁺ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg²⁺ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.