Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen biosynthesis

It has been speculated that heart valve interstitial cells (VICs) maintain valvular tissue homeostasis through regulated extracellular matrix (primarily collagen) biosynthesis. VICs appear to be phenotypically plastic, inasmuch as they transdifferentiate into myofibroblasts during valve development, disease, and remodeling. Under normal physiological conditions, transvalvular pressures (TVPs) on the right and left side of the heart are vastly different. Hence, we hypothesize that higher left-side TVPs impose larger local tissue stress on VICs, which increases their stiffness through cytoskeletal composition, and that this relation affects collagen biosynthesis. To evaluate this hypothesis, isolated ovine VICs from the four heart valves were subjected to micropipette aspiration to assess cellular stiffness, and cytoskeletal composition and collagen biosynthesis were quantified by using the surrogates smooth muscle alpha-actin (SMA) and heat shock protein 47 (HSP47), respectively. VICs from the aortic and mitral valves were significantly stiffer (P < 0.001) than those from the pulmonary and tricuspid valves. Left-side isolated VICs contained significantly more (P < 0.001) SMA and HSP47 than right-side VICs. Mean VIC stiffness correlated well (r = 0.973) with TVP; SMA and HSP47 also correlated well (r = 0.996) with one another. Assays were repeated for VICs in situ, and, as with in vitro results, left-side VIC protein levels were significantly greater (P < 0.05). These findings suggest that VICs respond to local tissue stress by altering cellular stiffness (through SMA content) and collagen biosynthesis. This functional VIC stress-dependent biosynthetic relation may be crucial in maintaining valvular tissue homeostasis and also prove useful in understanding valvular pathologies.     

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

Lipase-catalyzed synthesis of sorbitol-fatty acid esters at extremely high substrate concentrations

Lipase-catalyzed synthesis of sorbitol-fatty acid esters was performed in eutectic media with extremely high substrate concentrations. Homogeneous eutectic melts of sorbitol and fatty acids of C6-C16 were prepared using an adjuvant mixture. Enhanced homogeneity of mixtures was confirmed by X-ray diffraction analysis. The substrate concentration was 3.63-6.67 M in the eutectic media, whereas in organic media the concentration was below 0.10 M. Esters were synthesized with an immobilized Candida antarctica lipase, and optimum conditions were analyzed. Compared to reactions in organic media, the initial reaction rate of ester synthesis and the overall productivity were significantly enhanced in eutectic media while the conversion yields were similar. Based on the kinetic analysis, highly viscous eutectic media were shown to influence the initial reaction rate and the apparent activation energy resulting in diffusion limitations.     

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST–Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracelluar secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.     

Genetic Polymorphism of Interferon-γ, Interferon-γ Receptor, and Interferon Regulatory Factor-1 Genes in Patients with Hepatitis B Virus Infection

The natural history of hepatitis B virus (HBV) infection is probably related to host immune factors. Interferon-γ (IFN-γ) plays significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of IFN-γ, IFN-γ receptor (IFNGR)-1 and 2, and interferon regulatory factor (IRF)-1 genes. Between March 2002 and December 2002, 614 Korean patients were enrolled in two different groups: an HBV clearance group (n = 201), who were hepatitis B surface antigen (HBsAg) negative with antibodies to HBsAg and hepatitis B core antigen, and an HBV persistence group (n = 413), who were repeatedly HBsAg positive. We assessed polymorphisms in the IFN-γ gene at position +874, in the IFNGR-1 gene at positions −56 and +95, in the IFNGR-2 gene at the second position of codon 64 (Gln64Arg), and in the IRF-1 gene promoter (−410, −388), and the genotype distributions of the HBV clearance and persistence groups were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with the IFN-γ, IFNGR-1 and 2, and IRF-1 gene polymorphisms under the codominant, dominant, and recessive models.     

Quantitative analysis of representative proteome components and clustering of Helicobacter pylori clinical strains

BACKGROUND: Several Helicobacter pylori proteins have been reported to be associated with severe symptoms of gastric disease. However, expression levels of most of these disease-associated proteins require further evaluation in order to clarify their relationships with gastric disease patterns. Representative proteome components of 71 clinical isolates of H. pylori were analyzed quantitatively to determine whether the protein expression levels were associated with gastric diseases and to cluster clinical isolates. METHODS: After two-dimensional electrophoresis (2-DE) of H. pylori isolates, spot intensities were analyzed using pdquest 2-D Gel Analysis Software. The intensities of 10 representative protein spots, identified by peptide fingerprinting using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using quadrupole TOF MS, were subjected to the nonparametric Mann-Whitney test and hierarchical agglomerative cluster analysis. The relationship between clusters and gastric diseases was analyzed by the chi-squared test. RESULTS: Although the spot intensities of the 10 representative proteins were highly variable within each gastric disease group, the expression levels of CagA, UreB, GroEL, EF-Tu, EF-P, TagD, and FldA showed some significant differences among the gastric disease patterns. On the basis of the 10 target protein intensities, hierarchical agglomerative cluster analysis generated a dendrogram with clusters indicative of chronic gastritis/gastric cancers and gastric/duodenal ulcers. CONCLUSION: These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.