Gelatin scaffold ameliorates proliferation & stem cell differentiation into the hepatic like cell and support liver regeneration in partial-hepatectomized mice model.

Stem cell-based tissue engineering is an emerging tool for developing functional tissues of choice. To understand pluripotency and hepatic differentiation of mouse embryonic stem cells (mESCs) on a three-dimensional (3D) scaffold, we established an efficient approach for generating hepatocyte-like cells (HLCs) from hepatoblast cells. We developed porous and biodegradable scaffold, which was stimulated with exogenous growth factors and investigated stemness and differentiation capacity of mESCs into HLCs on the scaffoldin-vitro. In animal studies, we had cultured mESCs-derived hepatoblast-like cells on the scaffold and then, transplanted them into the partially hepatectomized C57BL/6 male mice model to evaluate the effect of gelatin scaffold on hepatic regeneration. The 3D culture system allowed maintenance of stemness properties in mESCs. The step-wise induction of mESCs with differentiation factors leads to the formation of HLCs and expressed liver-specific genes, including albumin, hepatocyte nucleic factor 4 alpha, and cytokeratin 18. In addition, cells also expressed Ki67, indicating cells are proliferating. The secretome showed expression of albumin, urea, creatinine, alanine transaminase, and aspartate aminotransferase. However, the volume of the excised liver which aids regeneration has not been studied. Our results indicate that hepatoblast cells on the scaffold implanted in PH mouse indicates that these cells efficiently differentiate into HLCs and cholangiocytes, forming hepatic lobules with central and portal veins, and bile duct-like structures with neovascularization. The gelatin scaffold provides an efficient microenvironment for liver differentiation and regeneration bothin-vitroandin-vivo. These hepatoblasts cells would be a valuable source for 3D liver tissue engineering/transplantation in liver diseases.

Receptor binding specificity of recent human H3N2 influenza viruses.

BACKGROUND: Human influenza viruses are known to bind to sialic acid linked alpha2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection. RESULTS: Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of alpha2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for alpha2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of alpha2-6 and alpha2-3 sialic acids, including human A549 and HeLa cells with high levels of alpha2-6 sialic acid, and CHO cells that have only alpha2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells. CONCLUSION: Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of alpha2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of alpha2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with alpha2-6 receptor specificity.

Mutation of two intramembrane polar residues conserved within the hyaluronan synthase family alters hyaluronan product size.

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHASE327Q and seHASE327K were expressed at low levels, whereas seHASE327D and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHASE327K,K48E protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (approximately 0.6 MDa) was made by seHASE327K,K48E and seHASK48E. The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses.

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via alpha2,6 (human) or alpha2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both alpha2,3- and alpha2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human alpha2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched alpha2,6 sialic acid or alpha2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.

Amount and avidity of serum antibodies against native glycoproteins and denatured virus after repeated influenza whole-virus vaccination.

There is still uncertainty on the correlates of protection by influenza vaccine. To determine the relationship between hemagglutination-inhibition (HI) titer and the specificity and avidity of serum antibodies, we analyzed serum from a longitudinal trial (1983-1987) of influenza vaccine efficacy [Keitel WA, Cate TR, Couch RB, Huggins LL, Hess KR. Efficacy of repeated annual immunization with inactivated influenza virus vaccines over a five year period. Vaccine 1997;15(10):1114-22 ]. We captured native virus particles with fetuin and separately measured relative antibody levels and avidities of antibodies against native glycoproteins and antibodies against denatured viral proteins. Most subjects had pre-existing antibodies against A/Victoria/75 and, although 70% had >two-fold increased antibodies against A/Philippines/82 after vaccination, only 30% showed increased antibodies to A/Victoria/75 indicating no dominance of original antigenic sin. There was variation in the levels of antibodies to unfolded antigens compared to native, but antibodies against denatured proteins never exceeded those against native virus. In some cases, the avidity increased without a significant increase in antibody concentration, which might explain why some vaccinees with low HI titer demonstrate adequate protection. We found that the negative correlation between pre-vaccination HI titer and the increase after vaccination is also seen when antibodies are measured directly, but that there is little relationship between HI titer and antibodies against native glycoproteins, either in amount or avidity. Our assay, which has also been adapted for recent influenza viruses that do not bind to fetuin, may be useful for vaccine evaluation.

Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase.

The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.

Inhibition of hyaluronan synthesis in Streptococcus equi FM100 by 4-methylumbelliferone.

As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4-methylumbelliferone (MU), although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane-rich fraction as an enzyme source in a cell-free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose-dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU-treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.

The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents, but conserved cysteine residues are not essential for enzyme function.

Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcUA) and UDP-GlcNAc to synthesize HA. The HAS from Streptococcus pyogenes (spHAS, 419 amino acids) contains six Cys residues, whereas the enzyme from Streptococcus equisimilis (seHAS, 417 amino acids) contains four Cys residues. These Cys residues of seHAS are highly conserved in all Class I HAS family members. Here we investigated the structural and functional roles of these conserved cysteines in seHAS by using site-directed mutagenesis and sensitivity to sulfhydryl modifying reagents. Both seHAS and spHAS were inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and iodoacetamide in a dose-dependent and time-dependent manner. These inhibition curves were biphasic, indicating the presence of sensitive and insensitive components. After treatment of seHAS with NEM, the V(max) value was decreased approximately 50%, and the K(m) values changed only slightly. All the Cys-to-Ala mutants of seHAS were partially active. The least active single (C226A), double (C226A,C262A), or triple (C226A,C262A,C367A) Cys mutants retained 24, 3.2, and 1.4% activity, respectively, compared with wild-type enzyme. Surprisingly, the V(max) value of the seHAS(cys-null) mutant was approximately 17% of wild-type, although the K(m) values for both substrates were increased 3-6-fold. Cys residues, therefore, are not involved in a critical interaction necessary for either substrate binding or catalysis. However, the distribution of HA products was shifted to a smaller size in approximately 25% of the seHAS Cys mutants, particularly the triple mutants. Mass spectroscopic analysis of wild-type and Cys-null seHAS as well as the labeling of all double Cys-to-Ala mutants with [(14)C]NEM demonstrated that seHAS contains no disulfide bonds. We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS.