Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

Measurement of hydroxyl radical (*OH) in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because generation of *OH is believed to be one of the major triggers of radiation injury. In this study, *OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of *OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of alpha-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN-methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of *OH but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was *OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of *OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.     

Comparison of vildagliptin twice daily vs. sitagliptin once daily using continuous glucose monitoring (CGM): Crossover pilot study (J-VICTORIA study)

Background

The role of Lipoprotein (a) cholesterol {Lp(a)-C}as an additional and/or independent risk factor for cardiovascular disease (CVD) is not clear. We evaluated the associations between Lp(a)-C and other CVD risk factors including plasma lipoprotein concentrations and body fatness in overweight and obese African American children.

Methods

A cross-sectional analysis was carried out using data from a sample of 121 African American children aged 9-11 years with Body Mass Index (BMI)'s greater than the 85th percentile. Body height, weight and waist circumference (WC) were measured. Fasting plasma concentrations of Lp(a)-C, Total cholesterol (TC), High density lipoprotein cholesterol (HDL-C), Very low density lipoprotein cholesterol (VLDL-C), Intermediate density lipoprotein cholesterol (IDL-C), Low density lipoprotein cholesterol (LDL-C), and Triacylglycerides (TAG) were analyzed using the vertical auto profile (VAP) cholesterol method.

Results

After adjusting for child age, gender, and pubertal status, Lp(a)-C was positively associated with both HDL-C and TC, and negatively associated with VLDL-C and TAG. Including BMIz and WC as additional covariates did not alter the direction of the relationships between Lp(a)-C and the other lipoproteins. Finally, after adjusting for the other plasma lipoproteins, Lp(a)-C remained strongly associated with HDL-C, whereas the associations of Lp(a)-C with the other lipoproteins were not significant when HDL-C was simultaneously included in the regression models.

Conclusions

Lp(a)-C was positively associated with HDL-C and this association is not influenced by other lipoprotein subclasses or by the degree of obesity. We conclude that Lp(a) cholesterol is not an independent risk factor for CVD in African American children.     

A brief review of bone adaptation to unloading

Weight-bearing bone is constantly adapting its structure and function to mechanical environments. Loading through routine exercises stimulates bone formation and prevents bone loss, but unloading through bed rest and cast immobilization as well as exposure to weightlessness during spaceflight reduces its mass and strength. In order to elucidate the mechanism underlying unloading-driven bone adaptation, ground-based in vitro and in vivo analyses have been conducted using rotating cell culturing and hindlimb suspension. Focusing on gene expression studies in osteoblasts and hindlimb suspension studies, this minireview introduces our recent understanding on bone homeostasis under weightlessness in space. Most of the existing data indicate that unloading has the opposite effects to loading through common signaling pathways. However, a question remains as to whether any pathway unique to unloading （and not to loading） may exist.     

Cytoplasmic domain of influenza B virus BM2 protein plays critical roles in production of infectious virus

Influenza B virus BM2 is a type III integral membrane protein that displays H⁺ ion channel activity. Analysis of BM2 knockout mutants has suggested that this protein is a necessary component for the capture of M1-viral ribonucleoprotein (vRNP) complex at the plasma membrane and for incorporation of vRNP complex into the virion during the assembly process. BM2 comprises 109 amino acid residues and possesses a longer cytoplasmic domain than the other 3 integral membrane proteins (hemagglutinin, neuraminidase, and NB). To explore whether the cytoplasmic domain of BM2 is important for infectious virus production, a series of BM2 deletion mutants lacking three to nine amino acid residues at the carboxyl terminus, BM2Δ107-109, BM2Δ104-109, and BM2Δ101-109, was generated by reverse genetics. Intracellular transport and incorporation into virions were indistinguishable between truncated BM2 proteins and wild-type BM2. The BM2Δ107-109 mutant produced levels of infectious virus similar to those of wild-type virus and displayed a spherical shape. However, the BM2Δ104-109 and BM2Δ101-109 mutants produced viruses containing dramatically reduced vRNP complex, as with BM2 knockout mutants, and formed enlarged, irregularly shaped virions. Moreover, gradient separation of membranes indicated that membrane association of M1 from mutants was greatly affected by carboxyl-terminal truncations of BM2. Studies of alanine substitution mutants further suggested that amino acid sequences in the 98-109 region are variable while those in the 86-97 region are a prerequisite for innate BM2 function. These results indicate that the cytoplasmic domain of the BM2 protein is required for firm association of the M1 protein with lipid membranes, vRNP complex incorporation into virions, and virion morphology.