Semi-automated method for the determination of free fatty acids in plasma

A semi-automated method for the determination of free fatty acids in plasma based on Mosinger's manual method (Mosinger, 1965) is described and the results of a comparison with Dole's titrimetric method (Dole, 1956) are presented. There is almost perfect correlation between the two methods, and the automated technique is simple, reliable, and significantly more accurate and precise than the titrimetric method.     

In Vivo Complementation of ureB Restores the Ability of Helicobacter pylori To Colonize

The objective of this study was to determine (i) if complementation of ureB-negative Helicobacter pylori restores colonization and (ii) if urease is a useful reporter for promoter activity in vivo. Strains used were M6, M6DeltaureB, and 10 recombinant derivatives of M6 or M6DeltaureB in which urease expression was under the control of different H. pylori promoters. Mice were orally inoculated with either the wild type or one of the mutant strains, and colonization, in vivo urease activity, and extent of gastritis were determined. Of eight M6DeltaureB recombinants tested, four colonized mice. Of those, three had the highest in vitro urease activity of any of the recombinants, significantly different from that of the noncolonizing mutants. The fourth colonizing recombinant, with ureB under control of the cag-15 promoter, had in vitro urease activity which did not differ significantly from the noncolonizing strains. In vivo, urease activities of the four colonizing transformants and the wild-type control were indistinguishable. There were no differences in gastritis or epithelial lesions between mice infected with M6 and those infected with the transformants. These results demonstrate that recovery of urease activity can restore colonizing ability to urease-negative H. pylori. They also suggest that cag-15 is upregulated in vivo, as was previously suggested by demonstrating that it is upregulated upon contact with epithelial cells. Finally, our results suggest that total urease activity and colonization density do not contribute to gastritis due to H. pylori.     

A tctex1-Ca2+ channel complex for selective surface expression of Ca2+ channels in neurons

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.     

Stage, survival and delays in lung, colorectal, prostate and ovarian cancer: comparison between diagnostic routes

BACKGROUND: Very few studies have reported cancer outcomes of patients referred through different routes, despite the prominence of current UK cancer urgent referral guidance. AIM: This study aimed to compare outcomes of cancer patients referred through the urgent referral guidance with those who were not, with respect to stage at diagnosis, survival, and delays in diagnosis. Design of study: Analysis of hospital records. SETTING: One hospital trust in England. METHOD: The records of 889 patients diagnosed in 2000-2001 with one of four types of cancer were analysed: 409 with lung cancer; 239 with colorectal cancer; 146 with prostate cancer; and 95 with ovarian cancer. Outcome measures were diagnostic stage, survival, referral and secondary care delays. RESULTS: For lung cancer, urgent referrals had more advanced TNM (tumor, node, metastasis) stage than patients diagnosed through other routes (P = 0.035) and poorer survival (P = 0.020). There was no difference in stage or survival for the other cancers. For each cancer, a higher proportion of urgent referrals was seen within 2 weeks. Secondary care delays for lung and colorectal cancer were shorter for inter-specialty referrals. CONCLUSION: For patients with lung cancer, the guidance appears to be prioritising those in the more advanced stages of disease. This was not the case for the other three cancers. Referral delays were shorter for patients urgently referred, as is the intention of the guidance. The avoidance of delays in outpatient diagnostics probably accounts for shorter secondary care delays for inter-specialty referrals.     

Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.     

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

Polymorphisms in the methylenetetrahydrofolate reductase and methionine synthase reductase genes and homocysteine levels in Brazilian children

Hyperhomocysteinemia is a risk factor for thrombosis, and methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms, folate, and B12 levels could contribute to plasma homocysteine (Hcy) variation. Although well established in adults, few studies have been performed in childhood. In this study, we investigated association of polymorphisms C677T and A1298C in the MTHFR gene and A66G in the MTRR gene with Hcy levels in children. These polymorphisms, as well as Hcy, folate, and vitamin B12 levels were investigated in 220 normal children with ages ranging from 1 to 8 years. Plasma Hcy, folate, and vitamin B12 levels were normal in all children. None of the polymorphisms could be considered an independent risk factor for hyperhomocysteinemia during childhood. The median Hcy levels in 37 children (17%) doubly heterozygous for C677T and A1298C mutations in the MTHFR gene were not different from the other genotypes. However, the association of the different genotypes with Hcy, folate, and vitamin B12 levels demonstrated significant P-values. The folate levels demonstrated a statistically significant decrease (P = 0.0477) from the C677T mutation in the MTHFR gene (TT genotype) when compared to the other groups. Folate was the only independent risk factor for hyperhomocysteinemia. Thus, monitoring the concentrations of folate would be more helpful for evaluating hyperhomocysteinemia and for preventing cardiovascular disease.     

Differentiation of the U-937 promonocytic cell line induced by phorbol myristate acetate or retinoic acid: Effect of aurothiomalate

Tissue macrophages, which participate in chronic synovial inflammation, differentiate from haemopoietic precursors in bone marrow and subsequently in tissue. During this process, they acquire attributes which are essential for their function in inflammation. Modulation of this process may represent a means of regulating inflammatory competence of macrophages in inflammatory joint disease. The action of aurothiomalate (ATM), an anti-rheumatic gold compound, on the differentiation of a promonocytic cell line (U937) was, therefore, examined inin vitro systems. U937 cells exposed to retinoic acid (RA) for 4 days or to phorbol myristate acetate (PMA) for 2 days acquired characteristics of macrophages, including the capacity to produce superoxide (O ₂ ^– ), responsiveness to formyl-methionyl-leucyl-phenylalanine (fMLP) and reduced proliferation. The activity of transglutaminase also increased in RA-exposed cultures. The effect of ATM exposure on acquisition of these characteristics was small and differed between RA- and PMA-stimulated cells.