Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitive M. bovis (BCG) and M. tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M. tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.     

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.     

Molecular genetic studies of sporadic pituitary tumors

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.     

Synergistic effects of relaxation and susceptibility in differentiation between compartmentalized and noncompartmentalized tissues

RATIONALE AND OBJECTIVES: This study illustrates the synergistic effects of relaxation- and susceptibility-based contrast enhancement. Using a combination of gadolinium (Gd) and dysprosium (Dy) complexes and a sequence capable of taking advantage of the particular relaxation behavior of this combination, the difference between compartmentalized and noncompartmentalized regions was significantly enhanced. METHODS: Magnetic resonance imaging of the rat kidney was performed before and immediately after the administration of a combination of Gd and Dy chelates (Gd-DTPA-BMA and Dy-DTPA-BMA). RESULTS: The signal intensity (SI) of the renal parenchyma was reduced by 85%, whereas the collecting tubes had a 100% increase of their SI as demonstrated by a short repetition time (600 msec), long echo time (50 msec), and spin-echo sequence. CONCLUSIONS: The high R2* effect, specific to the compartmentalized tissues, associated with the moderately high R1 and R2 developed in the remaining areas, results in an important improvement in tissue differentiation, which potentially is useful for the evaluation of pathological changes as in tubular necrosis.     

Reduced COX-2 protein in colorectal cancer with defective mismatch repair

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.     

Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor alpha and interleukin-1 in rheumatoid arthritis

OBJECTIVE: To investigate the regulation of expression of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA), in order to determine whether new blood vessel formation could be a potential therapeutic target in RA. METHODS: Dissociated RA synovial membrane cells were cultured in the presence of cytokine inhibitors, or under hypoxic conditions. Serum VEGF levels were serially measured in RA patients enrolled in clinical trials of anti-tumor necrosis factor alpha (anti-TNFalpha) monoclonal antibody treatment. RESULTS: Combined neutralization of TNFalpha and interleukin-1 (IL-1) in RA synovial membrane cultures reduced VEGF release by 45% (P < 0.05 versus control), although blockade of either TNFalpha or IL-1 activities alone resulted in only small inhibitory effects. In addition, release of VEGF from RA synovial membrane cells was selectively up-regulated by hypoxia. Serum VEGF levels were significantly elevated in RA patients relative to control subjects, and correlated with disease activity. Treatment of RA patients with anti-TNFalpha significantly decreased serum VEGF, and this effect was enhanced by cotreatment with methotrexate. CONCLUSION: Inhibition of TNFalpha and IL-1 activity in vivo could reduce the drive to new blood vessel formation, and hence pannus mass, adding to other therapeutic effects of anti-TNFalpha therapy in RA.     

Circadian changes in levels of free amino acids in rat retina, and regulation of them by melatonin

Alpha-tocopherol (vitamin E) is an important fat-soluble antioxidant in biological systems and, as a result of scavenging reactive oxygen, it is converted to alpha-tocopherylquinone. Alpha-tocopherol binds to alpha-tocopherol transfer protein (alphaTTP) in the liver cytosol, whereas alpha-tocopherylquinone does not. We found that alpha-tocopherylquinone binds to a liver protein with a molecular mass of about 40 kDa that is distinct from alphaTTP. This alpha-tocopherylquinone binding protein was purified further by multiple-step column chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the final preparation yielded a single band with an apparent molecular mass of 25 kDa, which microsequencing revealed was identical to glutathione-S-transferase (GST). The GST activity was inhibited in the presence of alpha-tocopherylquinone, as it is by other non-substrate ligands for GST, confirming that GST and alpha-tocopherylquinone interact directly. Alpha-tocopherylquinone binds to GST and may be transported to the site of metabolism or excreted in the bile as other non-substrate ligands for GST.     

Can reduction in hypertriglyceridaemia slow progression of microalbuminuria in patients with non-insulin-dependent diabetes mellitus?

The objective of this study was to investigate whether reduction in hypertriglyceridaemia is associated with a slower rate of progression of microalbuminuria in patients with non-insulin-dependent diabetes mellitus (NIDDM). Fifteen normotensive NIDDM patients with hypertriglyceridaemia (> 2.5 mmol L-1) and microalbuminuria were randomly selected to receive either placebo (eight patients) or gemfibrozil 600 mg b.i.d. (seven patients). Progression of microalbuminuria was assessed during a 12-month follow-up period with measurements, consisting of blood tests and triplicate 24-h urine collections, at 1, 3, 6, 9 and 12 months. All but one patient in the treatment group showed a favourable response (> or = 20% reduction) of hypertriglyceridaemia to gemfibrozil. One patient in the placebo group showed a spontaneous reduction in triglyceride levels. Progression of microalbuminuria was lower, although not statistically significantly so, in the treatment group (36%) than in the placebo group (65%). In the group with > or = 20% reduction in triglyceride levels, progression of MA was significantly lower than in the group with stable or increasing triglyceride levels (+1%, range -56% to +49% vs. +97%, range -35% to +202% respectively) (P = 0.03). Continued follow-up data of patients switching from placebo to gemfibrozil after the trial further support the role of serum triglyceride reduction in stabilizing albumin excretion. In conclusion, the results indicate that, in microalbuminuric NIDDM patients, effective treatment of dyslipidaemia could be associated with stabilization of urinary albumin excretion.     

Patch clamp detection in capillary electrophoresis

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.