To investigate RCL2 as a fixative for tissue fixation in routine histopathological examination and to assess tissue suitability for ancillary investigations.
The typical concentration of protein loaded varies from 0.13 to 1.40 μg/μL for a classical silver staining method in 2DE gel. Here, we present a simple modified classical silver staining method by modifying the silver impregnation and development reaction steps. This modified method detects the protein spots at extremely low loaded concentrations, ranging from 0.0048 to 0.0480 μg/μL. We recommend this modified silver staining as an excellent method for the limited biological samples used for silver-stained 2DE analysis. Altogether, the protocol takes close to two days from first dimension separation to second dimension separation, followed by silver staining, scanning, and analysis.
A tourist was infected with a new strain of leptospires namely, Leptospira icterohemorrhagiae serovar Lai strain Langkawi, when he was on vacation in Langkawi, Malaysia. The leptospiral strain was successfully isolated from the patient in the Netherland. In this study, the bacteria were retrieved from Holland and inoculated into fifteen guinea pigs in Universiti Putra Malaysia (UPM) to determine its pathogenicity. The main clinical symptoms in the guinea pigs were decreased appetite and jaundice. Blood profile showed high neutrophil, lymphocyte, PCV, RBC, haemoglobin, leukocyte and thrombocyte counts. Besides that, enhancement of electrolytes such as sodium (Na), chloride (Cl), and potassium (K) was also noted. Biochemical examination showed an increase alkaline phosphatase (ALP), aspartate transaminase (AST) and bilirubin levels. Albumin, alanine transaminase (ALT), blood urea, total protein and creatinine were low values. Histopathological examination under haematoxylin and eosin staining showed evidence of haemorrhages, congestion and oedema in all organs, with inflammatory cell infiltration characterized by neutrophils, lymphocytes and macrophages. Hydropic degeneration and cell necrosis were also common in the findings. Leptospires were detected from Day 2 p.i by silver staining and transmission electron microscopy (TEM). Rise in antibody titre was seen as early as Day 5 p.i and leptospiral DNA was detected by PCR in the kidneys and liver on Day 3 and Day 5, respectively. The findings were indicative of leptospirosis. This study demonstrated that guinea pigs are a suitable animal model to illustrate the clinical symptoms and pathological changes seen following infection with Leptospira icterohaemorrhagiae serovar Lai strain Langkawi. In general, the symptoms and changes seen in leptospirosis are similar to viral infections and the information and data from this present study would help differentiate infection due to leptospires from that of viral infection. Leptospiral infection has often been misdiagnosed to be viral infection such as influenza and dengue which have similar signs and symptoms as leptospirosis.
Fifteen (15) guinea pigs were experimentally infected with Leptospira icterohemorrhagiae serovar Lai strain
Langkawi, a new strain that was isolated from a human leptospirosis patient. Hematoxylin and Eosin ((H&E) staining
showed haemorrhages, congestion and oedema in all internal organs examined (lungs, liver, spleen and kidneys) with
inflammatory cell infiltration characterized by neutrophils, lymphocytes and macrophages. Hydropic degeneration and
cell necrosis were also common in our findings. Leptospires were detected starting Day 2 p.i by silver staining and
Transmission Electron Microscopy (TEM). Rise in antibody titres started on Day 5 p.i and leptospiral DNA was
detected beginning Day 3 in the kidneys and Day 5 in the liver by Polymerase Chain Reaction (PCR) assay. The
findings illustrated the pathogenesis of leptospirosis in guinea pigs which disclosed them as a suitable animal model for
demonstration of clinical symptoms of leptospirosis and pathological changes after being infected with Leptospira
icterohaemorrhagiae serovar Lai strain Langkawi, particularly pulmonary haemorrhages, a leading cause of mortality
in human leptospirosis.
Pleurotus pulmonarius has been reported to have a potent remedial effect on diabetic property and considered to be an alternative for type 2 diabetes mellitus treatment. This study aimed to investigate the antidiabetic properties of ammonium sulphate precipitated protein fractions from P. pulmonarius basidiocarps. Preliminary results demonstrated that 30% (NH4)2SO4 precipitated fraction (F30) inhibited Saccharomyces cerevisiae α-glucosidase activity (24.18%), and 100% (NH4)2SO4 precipitated fraction (F100) inhibited porcine pancreatic α-amylase activity (41.80%). Following RP-HPLC purification, peak 3 from F30 fraction demonstrated inhibition towards α-glucosidase at the same time with meagre inhibition towards α-amylase activity. Characterisation of proteins using MALDI-TOF/TOF MS demonstrated the presence of four different proteins, which could be implicated in the regulation of blood glucose level via various mechanisms. Therefore, this study revealed the presence of four antidiabetic-related proteins which are profilin-like protein, glyceraldehyde-3-phosphate dehydrogenase-like protein, trehalose phosphorylase-like (TP-like) protein, and catalase-like protein. Hence, P. pulmonarius basidiocarps have high potential in lowering blood glucose level, reducing insulin resistance and vascular complications.
The objective of this study was to ascertain the extent changes have occurred in the epidemiology of human rotavirus electropherotypes from the same location 7 to 8 years after an earlier study. Genomic RNA profiles of rotaviruses from diarrhoeic children admitted to the Kuala Lumpur Hospital from April to December 1996 were determined by polyacrylamide gel electrophoresis and silver staining. A total of 179 group A rotaviruses were detected from 870 children: 175 with legible staining of all RNA segments were classified into 14 distinct electropherotypes (10 and 4 with long and short migration patterns respectively). In addition, the results revealed: high predominance of long pattern electropherotypes (94% of the total electropherotypes); most long electropherotypes with RNA profiles which all 11 RNAs migrated separately (8 of 10 electropherotypes); all short electropherotypes had segments 2 and 3 that co-migrated; presence of a very numerically dominant electropherotype (75% of all electropherotypes); frequent co-circulation of the dominant electropherotype-present throughout the study period--with other electropherotypes present for limited periods; sequential temporal appearances by similar electropherotypes. These observations were similar to that of an earlier study conducted in 1988/89. Nevertheless, the dominant electropherotype in the present study was different and not among the electropherotypes detected in the earlier study.
Fine needle aspiration (FNA) cytology is now an integral part of the pre-operative investigation of breast lesions and the therapeutic protocol is today often planned on the basis of cytodiagnosis. However, from time to time the cytological picture may be equivocal or inconclusive. In recent years, nucleolar organizer region (NOR) scores have been explored for potential value in the diagnosis of malignancy as the scores in malignant nuclei are seen to be higher than in benign or reactive nuclei. With a view to applying NOR scoring in the evaluation of cytologically equivocal cases, we adopted the argyrophil technique for staining NOR s (AgNOR) in FNA cytological smears of 56 breast lesions, comprising 31 benign and 25 malignant lesions. Histological correlation was possible in 26 of these cases (17 malignant and 9 benign) and AgNOR scoring was done on paraffin sections of these as well. There was a significant difference between mean AgNOR scores in benign and malignant lesions in the cytological smears (P < 0.001). The AgNOR scores ranged from 2.5 to 5.0 per cell in benign lesions and 5.8 to 17.2 per cell in malignant lesions. None of the cases fell into the gray zone of overlap. One malignant lesion that was cytologically equivocal showed a mean AgNOR score of 6.08. The AgNOR scores on histological sections also showed a statistically significant difference (P < 0.001) between benign and malignant lesions with mean scores ranging from 1.34 to 2.58 dots per cell in benign lesions and scores of 2.42 to 5.28 dots per cell in malignant lesions. However, the scores overlapped in four cases and therefore it was considered unsuitable for routine diagnostic work. From this preliminary study, we conclude that an FNA AgNOR score of 5.0 and less strongly favours a benign lesion whereas a score above 5.0 would be in favour of a malignant lesion. A larger study would be needed to verify our impression that AgNOR scoring can be useful in cytologically equivocal cases.
The expression of high-abundance serum proteins in newly diagnosed patients with endometrial adenocarcinoma (EACa), squamous cell cervical carcinoma (SCCa) and cervical adenocarcinoma (ACCa), relative to control female subjects, was analyzed by subjecting serum samples to 2-DE followed by image analysis of the silver-stained protein profiles. The three cohorts of cancer patients demonstrated different altered expression of serum high-abundance proteins compared to negative control women. The expression of alpha1-antitrypsin, alpha1-B glycoprotein, cleaved high-molecular-weight kininogen (light chain) and antithrombin III were consistently altered in all the patients. However, clusterin was upregulated only in the patients with EACa, while those with SCCa and ACCa were typically characterized by the upregulated expression of zinc alpha-2-glycoprotein. The aberrant expression of selective serum proteins in the various cohorts of cancer patients was validated by competitive ELISA as well as by lectin detection. Analysis by using the champedak galactose binding lectin further highlighted an unidentified protein that may be differently glycosylated in the sera of the EACa patients that were studied.