Papain-mediated reduction of MTT is an artifact but not an indicator of cell apoptosis in Saccharomyces cerevisiae

Papain is a proteolytic enzyme of the cysteine protease family used for tissue dissociation and cell separation. Papain’snonspecific proteolysis of the plasma membrane enzymes plays a crucial role in the homeostasis by disrupting theintracellular pH of the affected cells which might lead to cell death. When the Saccharomyces cerevisiae cellswere treated with different concentrations of papain (1.0, 5.0, 10.0, 20.0, and 30.0 µg/ml), we found no alterationin the trans-plasma membrane electron transport (TPMET) activity and the intracellular pH of the cells, while itsignificantly decreased the mitochondrial dehydrogenase activity when measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Additional verification of cell viability by trypan blue assay showed98%–99% cell viability, contrary to the higher cell death observed with MTT assay. To better understand the decreasein cell viability with MTT assay, we tested the cell-free system that demonstrated a significant decrease of MTTconcentration but the trypan blue assay showed more number of viable cells. This study shows that papain interfereswith the MTT assay.

Mismatch repair genes expression defects & association with clinicopathological characteristics in colorectal carcinoma.

Background & objectives: DNA mismatch repair gene (MMR) abnormalities are seen in 95 per cent of hereditary nonpolyposis colorectal cancer (HNPCC) and 10-15 per cent of sporadic colorectal cancers. There are no data on MMR abnormalities in Malaysian colorectal cancer patients. This study was aimed to determine the frequency of abnormal MMR gene protein expression in colorectal carcinoma in Northern Peninsular Malaysia using immunohistochemistry. Methods: Clinicopathological information was obtained from 148 patients’ records who underwent bowel resection for colorectal cancer (CRC) at the three hospitals in Malaysia. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins were performed on paraffin embedded tissue containing carcinoma. Results: A total of 148 subjects and 150 colorectal carcinomas of sporadic and hereditary types were assessed. Three patients had synchronous tumours. Twenty eight cancers (18.6%) from 26 subjects (17.6%) had absent immunohistochemical expression of any one of the MMR gene proteins. This comprised absent MLH1 only – 3 cancers, absent MSH2 only – 3, absent MSH6 only – 2, absent PMS2 only – 3, absent MLH1 and PMS2 – 14, absent MSH2 and MSH6 – 2 and absent MLH1, MSH6 and PMS2 – 1. There was significant association between abnormal MMR gene protein expression and proximal colon cancers, mucinous, signet ring and poorly differentiated morphology. Interpretation & conclusions: Cancers with abnormal MMR gene expression were associated with microsatellite instability-high (MSI-H) phenotype. About 15 per cent demonstrated absent MSH2, MSH6 and PMS2 protein expression in isolation or in combination with other MMR genes, which often predicts a germline mutation, synonymous with a diagnosis of HNPCC. This appears to be high frequency compared to reported data.

Methylthioadenosine phosphorylase expression in cutaneous squamous cell carcinoma.

The methylthioadenosine phosphorylase (MTAP) gene is a tumour suppressor gene, located on chromosome 9p21, 100 kb telomeric of the p15 and p16 genes, which are often deleted in tumor cells. The role of MTAP protein expression in the genesis of cutaneous squamous cell carcinoma (SCC) is currently not known. In a previous study we have shown the frequent occurrence of allelic imbalance/loss of heterozygosity (AI/LOH) in cutaneous SCCs using AI/LOH markers flanking the p15, p16, and MTAP genes and demonstrated reduction in p15 and p16 protein expression in comparison to normal human skin. The present study is a continuation to our previous studies, aimed at determining possible roles played by MTAP protein expression in the genesis of cutaneous SCC. The expression of MTAP protein was detected using immunohistochemical approach in 109 micro array cutaneous SCC and 20 normal human skin tissue samples. The expression of MTAP was not significantly different in the cutaneous SCC cases as compared with normal human skin. This may indicate that MTAP protein expression does not contribute to the genesis of cutaneous SCC.

Rapid diagnosis of Helicobacter pylori infection in gastric imprint smears.

The objective of this study was to determine the sensitivity, specificity, positive (PPV), and negative predictive values (NPV) of Diff-Quik-stained gastric imprint cytology smears in the detection of H. pylori compared with histology. Air-dried imprint smears of gastric biopsies from 150 patients were stained by the Diff-Quik method in the endoscopy suite and examined for H. pylori, providing results within minutes. The presence of inflammation and intestinal metaplasia were documented. The same biopsy was processed and stained with H&E and Warthin-Starry stains, and reviewed by a different pathologist blind to the imprint cytology results. Ninety-four of the 150 patients were male with a mean age of 50 years. Based on histology, the H. pylori prevalence was very low at 8%. The sensitivity and specificity of imprint cytology in the detection of H. pylori were 83.3% and 100%, respectively. The PPV and NPV were 100% and 98.6%, respectively. There were two false negatives and no false positives. A combination of imprint cytology and histology achieved 100% sensitivity. Imprint smears did not provide added value over histology with regards to inflammation and metaplasia. Gastric imprint smears stained with Diff-Quik method is a rapid, cheap, and reliable method for the detection of H. pylori and have their best results when complemented with histology.

Trichuriasis: localized inflammatory responses in the colon.

Most patients with trichuriasis have light worm burdens. Data regarding the inflammatory response to Trichuris worms in the colon of lightly infected persons are scant. Nine patients whose Trichuris infection was found by colonoscopy had biopsies taken from a site adjacent to visible worms and from a second site some 20 cm distally. The biopsies were studied by routine and immunohistochemical methods. None of the biopsies showed mucosal ulceration, significant congestion, fibrosis, gland distortion or goblet cell mucin depletion. There was no difference between worm and worm-free sites in terms of edema, lymphoid follicles or epithelial slough. Worm sites had higher numbers of eosinophils, neutrophils and total inflammatory cells and lower numbers of plasma cells. However there was no difference in lymphocyte, mast cell, and B- and T-cell counts between the two sites. This suggests that the T. trichiura worm incites a local inflammatory response involving eosinophils and neutrophils, even when the colon has only a light burden of worms.