Partial characterization of an unusual 185 kDa protein synthesized by dermal fibroblasts from patients with Marfan syndrome: identification of the protein as type IV collagen

Production of an unusual collagenous protein was observed in culture of dermal fibroblasts from four patients with Marfan syndrome. The apparent molecular weight of the protein was about 185 kDa after reduction with 2-mercaptoethanol and 175 kDa after limited pepsin treatment. The 185 kDa protein was susceptible to the bacterial collagenase but resistant to the animal collagenase. Immunoprecipitation revealed the specific interaction of the pepsin-treated 175 kDa collagenous protein with monoclonal and polyclonal antibodies to human type IV collagen. From the patterns of CNBr peptide mapping the 185 kDa band was identified as alpha 1 (IV) chain. Type IV collagen in the skin is generally considered to be of non-fibroblastic origin. However, in "diseased" condition, dermal fibroblasts might produce type IV collagen. The clinical manifestation in relation to production of type IV collagen by cultured skin fibroblasts from Marfan patients is discussed.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or x-rays

Summary The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273^Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.     

Regioselective and enzymatic production of γ-resorcylic acid from resorcinol using recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells expressing a novel decarboxylase gene

A recombinant Escherichia coli, expressing the rdc gene, which encodes a γ-resorcylic acid decarboxylase (Rdc) reversibly catalyzing regioselective carboxylation of resorcinol derived from Rhizobium radiobacter WU-0108, converted 20 mM resorcinol to γ-resorcylic acid with a 44% (mol/mol) yield at 30°C for 7 h. The recombinant E. coli cells were recyclable at least five times for use as biocatalysts.     

Intestinal immunity suppresses carrying capacity of rats for the model tapeworm, Hymenolepis diminuta

Hymenolepis diminuta is a parasitic tapeworm of the rat small intestine and is recognized as a useful model for the analysis of cestode-host interactions. In this study, we analyzed factors affecting the biomass of the tapeworm through use of rat strains carrying genetic mutations, namely X-linked severe combined immunodeficiency (xscid; T, B and NK cells deficiency), nude (rnu; T cell deficiency), and mast cell deficient rats. The worm biomass of F344-xscid rats after infection with 5 cysticercoids was much larger than control F344 rats from 3 to 8?weeks. The biomass of F344-rnu rats was also larger than the controls, but was intermediate between F344-xscid and control rats. These observations demonstrated that host immunity can control the maximal tapeworm biomass, i.e., carrying capacity, of the rat small intestine. Both T cell and other immune cells (B and NK cells) have roles in determining the carrying capacity of tapeworms. Total worm biomass and worm numbers in mast cell deficient rats (WsRC-Ws/Ws) were not significantly different from control WsRC-+/+ rats after 3 and 6?weeks of primary infection. Mast cell deficient rats displayed reinfection resistance for worm biomass but not worm expulsion. These findings suggest that the mast cell has a role for controlling the biomass of this tapeworm in reinfection alone, but does not affect the rate of worm expulsion. Overall, our findings indicate that the mast cell is not a major effector cell for the control of the carrying capacity of tapeworms. The identity of the major effector cell remains unknown.     

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.     

A cyanophycin synthetase from <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> BP-1 catalyzes primer-independent cyanophycin synthesis

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.     

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a ‘cannabinoid-like’ substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.