Optimization and effect of dairy industrial waste as media components in the production of hyaluronic acid by Streptococcus thermophilus

Hyaluronic acid (HA) production using a dairy industrial waste is a more cost-efficient strategy than using an expensive synthetic medium. In this study, we investigated the production of HA using Streptococcus thermophilus under shake flask conditions using dairy industrial waste as nutritional supplements, namely whey permeate (WP) and whey protein hydrolysate (WPH). Preliminary screening using Plackett–Burman design exhibited WP, WPH, initial pH, and inoculum size as significant factors influencing HA titer. Response surface methodology design of four factors was formulated at three levels for enhanced production of HA. Shake flask HA fermentation by S. thermophilus was performed under global optimized process conditions and the optimal HA titer (342.93?mg?L^?1) corroborates with Box–Behnken design prediction. The molecular weight of HA was elucidated as 9.22–9.46?kDa. The ultralow-molecular weight HA reported in this study has a potential role in drug and gene delivery applications.     

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Laser light scattering measurements on vitreous and rooster comb hyaluronic acids

Molecular weights and translational diffusion coefficients have been measured for rooster comb and vitreous hyaluronic acid (HA) at pH 7.2 and 11. The results indicate that the molecular weight, second virial coefficient and translational diffusion coefficient for vitreous HA can be reversibly decreased by increasing the solution pH from 7.2 to 11, whereas the physical properties of rooster comb HA are independent of pH studied. In addition, it is reported that the second virial coefficient for vitreous HA is negative, suggesting intermolecular interactions exist in solution at both neutral and alkaline pH as opposed to rooster comb HA which exhibits a positive second vitrial coefficient associated with decreasing molecular weights may be related to the accessibility and number of hydrogen bond forming groups. Differences in the dependence of molecular weight on pH between vitreous and rooster comb HA may be due to differences in the number of intramolecular interactions per molecule. These studies indicate that molecules of low molecular weight HA are able to form higher molecular weight complexes and differences in the organization of the polysaccharide chains may contribute to the differences in molecular weight of HAs isolated from various tissues.     

Gas-liquid Chromatographic Determination of Carbohydrates in Tobacco

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight carbonyl compounds examined. The enzyme reaction on oxidized PVA resulted in a rapid decrease in viscosity, a fall of pH, and production of carboxylic acids. The enzyme, therefore, is considered to be an oxidized PVA hydrolase.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to an oxidized PVA hydrolase previously purified from another fraction adsorbed on SP-Sephadex at pH 7.0 from the PVA-degrading enzyme activities [Agric. Biol. Chem., 45, 63 (1981)]. The relations between these two oxidized PVA hydrolases are discussed.     

一株3-苯氧基苯甲酸降解菌的筛选及其协同Bacillus licheniformis G-04降解高效氯氰菊酯的研究

【目的】从长期受拟除虫菊酯类农药污染的白菜根系土壤分离1株3-苯氧基苯甲酸(3-phenoxybenzoic acid, 3-PBA)降解菌,并探究其与Bacillus licheniformis G-04协同作用对高效氯氰菊酯(beta-cypermethrin,Beta-CP)的降解及污染土壤的生物修复,为土壤农药残留危害处理提供优良菌种。【方法】采用富集驯化、筛选纯化方法,筛选3-PBA降解菌,并通过形态和生理生化特征以及16S rRNA序列分析进行鉴定。利用Origin 8.0分析3-PBA降解菌与B. licheniformis G-04的生长降解动力学过程。同时,采用高效液相色谱法评估两菌株协同降解Beta-CP的能力及其对受Beta-CP污染土壤的修复作用。【结果】筛选得到1株3-PBA高效降解菌HA516,48 h对3-PBA (100 mg/L)的降解率达到87.73%,经鉴定为皮特不动杆菌(Acinetobacter pittii);构建了该菌株和B. licheniformis G-04的生长降解动力学方程,结果表明模型与实验数据能较好拟合;以6.7∶3.3的接种比例先接种B. licheniformis G-04,24 h后再接入A. pittii HA516协同作用,在48 h,Beta-CP (50 mg/L)的降解率达78.37%,较单菌株(B. licheniformisG-04)的降解率(40.47%)提高了37.90%,半衰期从58.39h缩短为24.51h。土壤修复实验表明,第7天协同组对Beta-CP(30mg/kg)的降解率较单菌株提高了33.26%,达到79.27%。【结论】A.pittiiHA516是1株3-PBA高效降解菌,能与B. licheniformis G-04协同增效降解Beta-CP,可作为修复3-PBA或拟除虫菊酯类农药污染的优良微生物资源。     

Intra-articular hyaluronan (hyaluronic acid) and hylans for the treatment of osteoarthritis: mechanisms of action

Although the predominant mechanism of intra-articular hyaluronan (hyaluronic acid) (HA) and hylans for the treatment of pain associated with knee osteoarthritis (OA) is unknown, in vivo, in vitro, and clinical studies demonstrate various physiological effects of exogenous HA. HA can reduce nerve impulses and nerve sensitivity associated with the pain of OA. In experimental OA, this glycosaminoglycan has protective effects on cartilage, which may be mediated by its molecular and cellular effects observed in vitro. Exogenous HA enhances chondrocyte HA and proteoglycan synthesis, reduces the production and activity of proinflammatory mediators and matrix metalloproteinases, and alters the behavior of immune cells. Many of the physiological effects of exogenous HA may be a function of its molecular weight. Several physiological effects probably contribute to the mechanisms by which HA and hylans exert their clinical effects in knee OA.     

Ageing of added copper in bentonite without and with humic acid

Abstract

The ageing of metals in soils plays an important role in assessing ecological risk and soil quality criteria, it therefore is necessary to understand the ageing processes. Using a sequential extraction procedure and a parabolic diffusion equation, the ageing of added copper(Cu) in bentonite without and with humic acid (HA) was investigated as a function of incubation time over one year. At the same time, the effect of pH on Cu ageing in bentonite with HA was also studied. The most labile Cu fractions (water-soluble Cu, Cu extracted by NH₄NO₃ and EDTA) gradually transformed into less extractable forms. The modelled diffusion equation suggested that the ageing processes were controlled by surface nucleation/precipitation and diffusion. In the presence of HA, the contribution from surface nuclea-tion/precipitation increased significantly, whereas that from diffusion decreased, this indicated the addition of HA significantly enhanced the short-term ageing by complexation with Cu ions, but strongly inhibited the further diffusion of Cu ions into interlayer of bentonite during long-term ageing owing to the more stable complexes formed on solid surface. With increasing pH in bentonite with HA, the apparent diffusion rate coefficient (D/r²) increased greatly, which showed that during long-term ageing pH played an important role in reducing the extractability as a result of the significant increase in residual Cu (RES-Cu), the most inert fraction, due to micropore diffusion.     

Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E₂ (PGE₂) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE₂ production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE₂, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE₂, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.     

Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy

BackgroundHemagglutinin (HA) of influenza A is one of the key virulence factors that mediates the release of viral components in host cells. HA is initially synthesized as a trimeric precursor (HA0) and then it is cleaved by proteases to become a functional HA. Low pH induces irreversible conformational changes in both HA0 and HA but only HA is fusion compatible. Here, we used high-speed atomic force microscopy (HS-AFM) to record conformational changes in HA0 trimers (H5N1) from neutral to acidic conditions at a millisecond scale.MethodsPurified HA0 protein was diluted with either neutral Tris-HCl (pH 7.4) or acetic acid-titrated Tris-HCl (pH 5.0) and then loaded onto bare mica. Neutral or acidic Tris-HCl was used as the scanning buffer. HS-AFM movies were recorded and processed using Image J software.ResultsThe conformation of HA0_neutral visualized using HS-AFM was comparable to the HA trimer structures depicted in the PDB data and the AFM simulator. HA0 underwent rapid conformational changes under low pH condition. The circularity and area of HA0_acid were significantly higher than in HA0_neutral. In contrast, the height of HA0_acid was significantly lower than in HA0_neutral.ConclusionsWe have captured real-time images of the native HA0 trimer structure under physiological conditions using HS-AFM. By analyzing the images, we confirm that HA0 trimer is sensitive to acidic conditions.General significanceThe dynamic nature of the HA structure, particularly in the host endosome, is essential for H5N1 infectivity. Understanding this acidic behavior is imperative for designing therapeutic strategies against H5N1. This article reports a sophisticated new tool for studying the spatiotemporal dynamics of the HA precursor protein.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Identification of Pt and Pd bound to humic acid species in spiked soils and street dusts by size exclusion chromatography and ICP-MS

Abstract

The formation of PGE-humic acid (HA) complexes in soil and street dust samples was investigated. In order to assess the distribution of Pt and Pd among molecular weight fractions of humic substances, the HA extracts (extracted by 0.1 mol L^?1 sodium pyrophosphate) were analysed by size exclusion chromato-graphy coupled on-line with UV-Vis detection. Similar chromatograms were obtained for soils and street dust samples (254 nm, 280 nm) and two size fractions were operationally defined as high (1600–5000 Da) and low molecular (< 1600 Da) HA fractions. The concentration of Pt and Pd in the separated extracts was determined by ICP-MS. The results indicate that up to 43% of Pt is in the high molecular and up to 52% in the low molecular HA fraction. In both type of soil samples, Pd is preferentially bound to the low molecular HA fraction. Dependence of Pd–HA and Pt–HA formation on the sample type both in the soils and in the street dust sample as well as on Pt oxidation state was established. Metallic Pt shows a tendency for complexation with the fractions of HA higher than 5000 Da. In the street dust samples, the distribution of Pt and Pd is similar and is strongly dependent on the sample type, being bound mainly to the fractions: higher than 5000 Da, 1600–5000 Da and the fraction lower than 1600 Da.     

Optimization of medium components for high-molecular-weight hyaluronic acid production by <Emphasis Type="Italic">Streptococcus</Emphasis> sp. ID9102 via a statistical approach

Hyaluronic acid (HA), linear high-molecular-weight glycosaminoglycan produced from Streptococcus sp., has raised interest in the medical and cosmetics industries because of the various biological functions of HA. In this paper, we report on the optimization of medium components for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) by two-step optimization (one-factor-at-a-time and taguchi orthogonal array design). In the first step, medium components, such as carbon, nitrogen, phosphate, and mineral sources, were selected for HA production in Streptococcus sp. ID9102 (KCTC 11935BP) using the one-factor-at-a-time method. In the second step, the concentration of the selected medium components was optimized using taguchi orthogonal array design. The design for medium optimization was developed and analyzed using MINITAB 14 software. In addition, the effect of amino acid and organic acid, such as glutamine, glutamate, and oxalic acid, was studied for HA production in Streptococcus sp. ID9102 (KCTC 11935BP). Through these processes, the optimum medium comprising 4% glucose, 0.75% yeast extract, 1.0% casein peptone, 0.25% K₂HPO₄, 0.05% MgCl₂, 0.5% NaCl, 0.04% glutamine, 0.06% glutamate, and 0.02% oxalic acid was determined. We were able to produce HA with a molecular weight of 5.9 × 10⁶ at a productivity of 6.94 g/l on pilot scale fermentation.     

Biodegradation of kraft lignin by a newly isolated bacterial strain, <Emphasis Type="Italic">Aneurinibacillus aneurinilyticus</Emphasis> from the sludge of a pulp paper mill

A kraft lignin-degrading bacterium (ITRC S ₇ ) was isolated from sludge of pulp and paper mill and characterized as Aneurinibacillus aneurinilyticus by biochemical tests and 16SrRNA gene sequencing. The bacterium did not utilize kraft lignin (KL) as the sole source of carbon and energy. However, this strain reduced the color (58%) and lignin content (43%) from kraft lignin-mineral salt medium when supplemented with glucose at pH 7.6 and 30°C after 6 days. The degradation on addition of glucose in culture medium is clear evidence of co-metabolism of KL by A. aneurinilyticus. The analysis of lignin degradation products by GC-MS in ethyl acetate extract from an A. aneurinilyticus-inoculated sample revealed the formation of low molecular weight aromatic compounds such as guaiacol, acetoguaiacone, gallic acid and ferulic acid, indicating that the bacterium can oxidize of the sinapylic (G units) and coniferylic (S units) alcohol units which are the basic moieties that build the hardwood lignin structure. The low molecular weight aromatic compounds identified in extracts of the inoculated sample favors the idea of biochemical modification of the KL to a single aromatic unit.     

降解尿酸乳酸菌复合菌系的筛选与菌株组合提升降解效果

【背景】高尿酸症由血液中尿酸含量明显升高而导致,利用乳酸菌对人体的益生作用缓解高尿酸血症越来越受到关注。【目的】获得具有降解尿酸能力的乳酸菌复合菌系与纯培养菌株。【方法】以泡菜为样品来源,以尿酸为底物,采用MRS培养基筛选降解尿酸的乳酸菌复合菌系,通过高效液相色谱法测定复合菌系对尿酸的降解能力。【结果】得到一组乳酸菌复合菌系,当培养温度为37 °C、pH值为6.20、静置培养72 h后复合菌系对尿酸的降解率为12.08%;通过优化培养条件,当该菌系在以牛肉膏为单一氮源、初始pH值为5.00、温度为35 °C的条件下培养72 h,尿酸降解率上升至17.19%,降解率比优化前提高了42.3%;从该菌系中分离出两株具有尿酸降解能力的菌株UA-1与UA-2,它们的尿酸降解率分别为10.85%和8.65%;通过形态学观察和16S rRNA基因序列分析,经鉴定两株菌均为布氏乳杆菌(Lactobacillus buchneri)。将两株单菌组合降解尿酸试验发现,UA-1与UA-2比例为2:1的尿酸降解率为20.2%,比原复合菌系的降解能力提高了67.22%。【结论】研究证明了乳酸菌复合菌系对尿酸的降解能力优于单个菌株,为后续利用乳酸菌复合菌系应用提供了数据支持。     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Investigation of hyaluronan function in the mouse through targeted mutagenesis

It has become increasingly apparent that the high molecular mass glycosaminoglycan, hyaluronan (HA), is required for many morphogenetic processes during vertebrate development. This renewed understanding of the various developmental roles for HA, has come about largely through the advent of gene targeting approaches in the mouse. To date, mutations have been engineered in the enzymes responsible for biosynthesis and degradation and for those proteins that bind to HA within the extracellular matrix and at the cell surface. Collectively, the phenotypes resulting from these mutations demonstrate that HA is critical for normal mammalian embryogenesis and for various processes in postnatal and adult life (Table 1). In this article we will review our progress in understanding the biological functions for HA through targeted mutagenesis of the HA synthase 2 (Has2) and 3 (Has3) genes. Data that has been obtained from a conventional targeted disruption of the Has2 gene, is presented in an accompanying review by Camenisch and McDonald. More specifically, in this review we will provide an overview of the conditional gene targeting strategy being used to create tissue-specific deficiencies in Has2 function, along with our progress in understanding the role for Has3-dependent HA biosynthesis. Published in 2003.     

State of aggregation of recombinant hirudin in solution under physiological conditions

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight ¯M _w determined by sedimentation equilibrium was found to be 6914±76 Da compared to 6964 Da expected from the amino acid sequence. The ¯M _z /¯M _w ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M _r ) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991),Eur. J. Biochem. 202, 67–73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konnoet al. (1988),Arch. Biochem. Biophys. 267, 158–166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.Abbreviations ¯M _w weight-average molecular weight - ¯M _z Z-average molecular weight - M _r relative molecular weight - NTSB 2-nitro-5-thiosulfobenzoic acid - Tris Tris(hydroxymethyl)aminomethane - r-HV1 recombinant desulfatohirudin - _M molar extinction coefficient