Localization of the feline sarcoma virus fgr gene product (P70gag-actin-fgr): association with the plasma membrane and detergent-insoluble matrix.

The v-fgr oncogene codes for a unique transforming protein (P70gag-actin-fgr) that contains virus-specific determinants and cell-derived sequences for both a tyrosine-specific kinase domain and an actin domain. We examined the subcellular distribution of the v-fgr protein by immunofluorescence microscopy and various cell fractionation techniques. By immunofluorescence, the v-fgr protein was localized in a diffuse cytoplasmic pattern within transformed cells. The v-fgr protein was not detectable at substratum adhesion sites. Crude membrane preparations (P100) obtained from fgr-transformed cells contained elevated levels of P70gag-actin-fgr. Further analysis of membranes on discontinous sucrose gradients revealed that P70gag-actin-fgr cofractionated with plasma membranes. Using an alternate method of fractionation, we found that the majority of the v-fgr protein remained with the insoluble matrix obtained by treating cells with a buffer containing Triton X-100. When membranes were similarly treated with detergent, nearly all of v-fgr protein remained with the residual insoluble matrix. These results suggest that the transforming activity of P70gag-actin-fgr may be directed to subcellular cytoskeletal targets at or near the cytoplasmic face of the plasma membrane.     

Effect of supplementall-tyrosine on pigment production in cultures of the Legionnaires' disease bacterium

The etiologic agent of Legionnaires' disease grows on certain agar media. Cultures of this organism on supplemented Mueller-Hinton agar are characterized by the appearance of brown pigment in and around areas of bacterial growth. The major peptone source in Mueller-Hinton agar is an acid hydrolysate of casein. Legionnaires' disease bacterium also grows on a medium in which the peptone source is 0.25% yeast extract, but no pigment is produced. If the yeast extract agar is enriched withl-tyrosine, pigment formation can occur. Pigmentation of cultures of Legionnaires' disease bacterium may be mediated by a phenolo-monooxygenase, or tyrosinase.     

Biopharmaceutical studies of spirobishexahydropyrimidine.

Oral administration of spirobishexahydropyrimidine showed an increase in the activity of serum transaminases, lactate dehydrogenase and alkaline phosphatase. Biological half life and other pharmacokinetic parameters showed rapid absorption and slow elimination of the drug.     

Biopharmaceutical studies of 3-substituted isatin derivatives

The metabolic fate of isatin hydrazone (Ia), isatin-3-thiosemicarbazone (Ib), isatin-3-semicarbazone (Ic), isatin-3-phenylhydrazone (Id), isatin oxime (Ie) and 3-hydroxy-3-acetonyl oxindole (II) was studied in rabbits. The compounds were administered orally in the dose of 300 mg/kg body wt. Isatin anthranilic acid, tryptophan and nicotinic acid were identified as the major metabolites excreted in urine. The 3-hydroxy-3-acetonyl oxindole (II) gave on additional metabolite, oxindole. The major metabolites were separated and identified unambiguously on thin layer silica gel plate. Metabolic pathways have been proposed to explain the biotransformation of the compounds investigated.     

Amphotropic host range of naturally occuring wild mouse leukemia viruses.

Seven murine leukemia virus field isolates (uncloned) from wild mice (Musmusculus) of four widely separated areas in southern California show an unusually wide in vitro host range. They replicate well in human, feline, canine, guinea pig, rabbit, rat, and mouse cells, whereas bovine, hamster, and avian cells are resistant. Since this host range includes that of both mouse tropic (ecotropic) and xenotropic murine leukemia viruses, they are designated as "amphotropic". No purely xenotropic virus component is detectable in these field isolates. They may represent the "wild" or ancestral viruses from which the ecotropic and xenotrophic murine leukemia virus strains of laboratory mice have been derived.     

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.     

Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice

ObjectivesTyphoid fever is caused by Salmonella enterica serovar Typhi. OmpC, OmpF and OmpA, the three major outer membrane proteins (OMPs), could serve as vaccine candidates.MethodsThe porins antigenicity was predicted in silico. The OMP genes were amplified, cloned and expressed. Sero-reactivities of the recombinant proteins purified by denaturing method were assayed by ELISA. BALB/c mice were immunized with the recombinant porins followed by bacterial challenge.ResultsBacterial challenge of the animal model brought about antibody triggering efficacy of the antigen in OmpF > OmpC > OmpA order. Experimental findings validated the in silico results. None of the antigens had synergic or antagonistic effects on each other from immune system induction points of view. Despite their high immunogenicity, none of the antigens was protective. However, administration of two or three antigens simultaneously resulted in retardation of lethal effect. Porins, in addition to their specific functions, share common functions. Hence, they can compensate for each other's functions.ConclusionsThe produced antibodies could not eliminate the pathogenicity by blockade of one or some of the antigens. Porin antigens are not suitable vaccine candidates alone or in denatured forms. Native forms of the antigens maybe studied for protective immunogenicity.     

Association of the lipoprotein receptor-related protein 2 gene with gout and non-additive interaction with alcohol consumption

Introduction

The T allele of a single nucleotide polymorphism (SNP: rs2544390) in lipoprotein receptor-related protein 2 (LRP2) is associated with higher serum urate and risk of gout in Japanese individuals. SNP rs2544390 also interacts with alcohol consumption in determining hyperuricemia in this population. We investigated the association of rs2544390 with gout, and interaction with all types of alcohol consumption in European and New Zealand (NZ) Māori and Pacific subjects, and a Māori study cohort from the East Coast region of NZ’s North Island.

Methods

Rs2544390 was genotyped by Taqman®. From NZ a total of 1205 controls and 1431 gout cases clinically ascertained were used. Publicly available genotype and serum urate data were utilized from the Atherosclerosis Risk in Communities (ARIC) study and the Framingham Heart Study (FHS). Alcohol consumption data were obtained by consumption frequency questions in all study cohorts. Multivariate adjusted logistic regression was done using STATA.

Results

The T allele of rs2544390 was associated with increased risk of gout in the combined Māori and Pacific Island cohort (OR = 1.20, P = 0.009), and associated with gout in the European subjects, but with a protective effect (OR = 0.79, P_Unadjusted = 0.02). Alcohol consumption was positively associated with risk of gout in Māori and Pacific subjects (0.2% increased risk/g/week, P = 0.004). There was a non-additive interaction between any alcohol intake and the risk of gout in the combined Māori and Pacific cohorts (P_Interaction = 0.001), where any alcohol intake was associated with a 4.18-fold increased risk in the CC genotype group (P = 6.6x10^-5), compared with a 1.14-fold increased risk in the CT/TT genotype group (P = 0.40). These effects were not observed in European subjects.

Conclusions

Association of the T-allele with gout risk in the Māori and Pacific subjects was consistent with this allele increasing serum urate in Japanese individuals. The non-additive interaction in the Māori and Pacific subjects showed that alcohol consumption over-rides any protective effect conferred by the CC genotype. Further exploration of the mechanism underlying this interaction should generate new understanding of the biological role of alcohol in gout, in addition to strengthening the evidence base for reduction of alcohol consumption in the management of gout.     

The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ₄₀, and the less common but more amyloidogenic form, Aβ₄₂. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ₄₂ amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ₄₂ amyloids. To further investigate the relationship between crocin and Aβ₄₂ structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ₄₂. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ₄₂. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.