Undiagnosed intraoperative methaemoglobinaemia.

Methaemoglobinaemia is a rare but potentially dangerous haemoglobinopathy that is often underdiagnosed. It is one of the causes for unexplained cyanosis with dark-coloured blood, especially in the absence of cardiac or pulmonary pathology. Not uncommonly so, it is an incidental perioperative finding in cases of dark-coloured blood not improving with oxygen in apparently acyanotic patients. The present case report is of a child with deaf-mutism posted for cochlear implant surgery who presented with 'chocolate-coloured blood' in the surgical field, despite blood gas analysis showing a normal partial pressure of oxygen.

Prevalence of aflatoxin B1 in liver biopsies of proven hepatocellular carcinoma in India determined by an in-house immunoperoxidase test.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death in the world. The incidence of HCC in India is reportedly low and varies from 0.2 to 1.9 %. Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, are potent human carcinogens implicated in HCC. The prevalence of aflatoxin B1 (AFB1) as co-carcinogen was analysed using an in-house immunoperoxidase test in 31 liver biopsies and 7 liver-resection specimens from histopathologically proven HCC, and in 15 liver biopsies from cirrhosis patients (control group). Serum was tested for hepatitis B and C serological markers using commercial assays, and for AFB1 using an in-house ELISA with a sensitivity of approximately 1 ng ml(-1) for AFB1. In spite of positive AFB1 immunostaining in HCC cases, all serum specimens, from both HCC and the control groups, were AFB1-negative. There were 18 (58.1 %) HCC cases that revealed AFB1 in liver biopsies; 68.8 % (n=11) of non-B non-C hepatitis cases with HCC and 46.1 % (n=6) of the hepatitis B surface-antigen-positive subjects were positive for AFB1. Out of the two hepatitis B/hepatitis C virus co-infected cases, one was positive for AFB1. Of seven tumour-resection samples, six were positive for AFB1. Only one case revealed AFB1 in the non-tumour area of the resected material. Thus AFB1 staining was significantly associated with tumour tissue (P=0.03). Aflatoxins proved to have a significant association with HCC in this peninsular part of the subcontinent. The impact seems to be a cumulative process, as revealed by the AFB1 deposits in HCC liver tissue, even though the serum levels were undetectable.

Angiogenesis in myelodysplastic syndromes (MDS) in Indian patients.

Angiogenesis plays an important role in the pathogenesis of haematological neoplasms and may be correlated with the prognosis. We recently evaluated the microvessel densities in trephine biopsy sections of seventeen patients of myelodysplastic syndromes (MDS). Of the 17 cases, 2 were RAEB-t, 3 were RAEB, one was RARS and 11 were of the subtype RA (FAB subtyping). The microvessel counts were measured in the bone marrow biopsy sections by immunohistochemical staining, using CD34 reactive monoclonal antibodies. MVD was significantly higher in the cases of RAEB and RAEB-t as compared to the cases of RA. The average MVD per x400 in the cases of RA was 5.7 +/- 4.7 with a median value of 4.65 (range 19) whereas it was 45.4 +/- 10.0 and 44.0 (range 27.3) respectively in RAEB and RAEB-t (p <.001), the 95% confidence interval being (2.94, 8.5) and (36.6, 54.3), for the two groups respectively. This finding may imply that subtypes of MDS with a higher tendency for converting to acute leukaemia are associated with increased angiogenesis as compared to other subtypes where the risk of progression to acute leukaemia is much lower.

Synthesis and antibacterial activity of various substituted s-triazines.

Series of substituted-s-triazines (1-22) were synthesized and evaluated for their in vitro antibacterial activity against six representative Gram-positive and Gram-negative bacterial strains. Many compounds have displayed comparable antibacterial activity against Bacillus sphaericus and significantly active against other tested organisms with reference to streptomycin.

Synthesis of multisubstituted quinolines from Baylis-Hillman adducts obtained from substituted 2-chloronicotinaldehydes and their antimicrobial activity.

Baylis-Hillman acetates were synthesized from substituted 2-chloronicotinaldehydes and were conveniently transformed into multisubstituted quinolines and cyclopenta[g]quinolines on reaction with nitroethane or ethyl cyanoacetate via a successive S(N)2'-S(N)Ar elimination strategy. Thus, synthesized quinolines were evaluated for antimicrobial activity and found having substantial antibacterial and antifungal activity.

Anti-malarial activity of Baylis-Hillman adducts from substituted 2-chloronicotinaldehydes.

New Baylis-Hillman adducts are synthesized based on substituted 2-chloronicotinaldehydes and screened for their in vitro anti-malarial activity against chloroquine sensitive and chloroquine resistant Plasmodium falciparum. Out of the six new compounds synthesized and screened, 2b, 2c and 2d compounds showed substantial anti-malarial activity.

Synthesis and antibacterial activity of 2,4,6-tri substituted s-triazines.

Various 2,4,6-tri substituted s-triazines were synthesized and screened for antibacterial activity against Gram-positive and Gram-negative organisms. These s-triazine derivatives displayed high in vitro antibacterial activities comparable to penicillin and streptomycin against tested microorganisms.

The expression of adhesion molecules in muscle biopsies: the LFA-1/VLA-4 ratio in polymyositis.

OBJECTIVES: The expression of three pairs of adhesion receptors and ligands was examined in 22 consecutive muscle biopsies showing morphological signs of inflammation. MATERIAL AND METHODS: The following groups were studied: patients with polymyositis (PM) (n=7), patients with myositis that did not fulfil criteria for PM, i.e. suspected PM (n=5), patients with other diseases, with no clinical signs of inflammatory myopathy (n=6), and a small group of non-PM inflammatory myopathies (n=4). The endothelial expression of ICAM-1, VCAM-1 and E-selectin was evaluated, as was the cellular expression of LFA-1, VLA-4 and SLex. In addition, the expression of MHC class I and II was studied. RESULTS: The ratio between the number of cells expressing LFA-1 and VLA-4 showed significant differences between the groups, with the lowest values in PM. CONCLUSION: The LFA-1/VLA-4 ratio should be suitable for diagnostic purposes. Our findings also indicate that the VLA-4/VCAM-1 system is important for chronic T cell inflammation in muscle, in line with findings in other "hidden" organs like joints and the central nervous system.

Curcumin-induced differentiation of mouse embryonal carcinoma PCC4 cells.

Curcumin, a natural component of turmeric extracted from the rhizomes of Curcuma longa, is known to exhibit a number of biological properties. In the present study, curcumin, at low concentration, was shown to induce differentiation in embryonal carcinoma cell line PCC4. In response to curcumin, PCC4 cells ceased to proliferate and showed cell cycle arrest at G1 phase after 4 hours of treatment, followed by their differentiation which is characterized by increase of nuclear/cytoplasmic ratio. The expression of hsp 70 was also seen upon 8 h of curcumin treatment, and it remained constant up to 48 h. Differentiated cells also expressed a series of differentiation markers such as lamin A, well-established actin, and keratin cytoskeleton. We used mRNA differential display analysis to identify the genes that are regulated during curcumin-induced differentiation of PCC4 cells. We cloned and sequenced three partial cDNAs that were differentially expressed in normal and differentiated cells. Sequence comparison of one downregulated cDNA (Al) has shown homology to a gene present on mouse chromosome five, while the two upregulated cDNA (C1 and C7) are homologous to several mouse ESTs clones from organs of mesodermal origin. We have identified the full-length coding sequence of the Cl fragment with a putative amino acid sequence. Tissue-specific Northern with RNA from adult mouse organs with the C1 fragment alone showed hybridization with mRNA from several tissues, whereas the same Northern with only the coding sequence showed expression of C1 gene mainly in the adult kidney. Homology search revealed that C1 sequence is part of the 3' UTR and may be common to several genes expressed in many tissues. Thus, curcumin appears to differentiate embryonal carcinoma cell PCC4, and one of the upregulated genes seems to be expressed mainly in the adult kidney.

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells.

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.

Diagnosis of hepatitis C virus infection by ELISA, RIBA and RT-PCR: a comparative evaluation.

OBJECTIVES: To evaluate the efficacy of second-generation ELISA (ELISA-2), third-generation ELISA (ELISA-3) and third-generation recombinant immunoblot assay (RIBA 3.0) for detection of antibodies to hepatitis C virus (anti-HCV) in comparison with reverse transcriptase-polymerase chain reaction (RT-PCR) to detect HCV RNA for the diagnosis of hepatitis C. METHODS: Sera of 108 patients with chronic liver disease (CLD) were analyzed by ELISA-2, ELISA-3, RIBA 3.0 and RT-PCR in the first part of the study; in the second part, sera of 105 patients with non-chronic liver disease were evaluated with ELISA-3, RIBA 3.0 and RT-PCR. RESULTS: In the CLD group, anti-HCV was positive in 4.6%, 14.8% and 16.6% by ELISA-2, ELISA-3 and RIBA 3.0, respectively. Among these anti-HCV positive cases, HCV RNA was positive in 100%, 58.9% and 64%, respectively. ELISA-2 did not give false-positive results, but missed substantial number of anti-HCV positive cases (p < 0.001). In the second group, anti-HCV was positive in 76.3% by ELISA-3 and 68.6% by RIBA 3.0 (p:ns). HCV-RNA was positive in 88.7% of ELISA- and RIBA-positive cases; in 60% of ELISA-positive, RIBA-indeterminate cases; and in 46.4% of ELISA-negative, RIBA-negative cases. CONCLUSIONS: ELISA-2 is not a suitable assay for routine screening. ELISA-3 was at par with RIBA 3.0 and it can be recommended for routine screening for anti-HCV. RT-PCR for HCV is of value in detecting early viremic, anti-HCV negative cases; this may be of importance in the treatment of hepatitis C.

Kinetics of platelet P-selectin mobilization: concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO donor SNAP.

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.

Staphylococcal enterotoxin-A-induced in-vitro adhesion of HL-60 cells to endothelial cells involves both selectin and integrin families of cell adhesion molecules.

In-vivo exposure to the bacterial superantigen Staphylococcal enterotoxin-A (SEA) induces an inflammatory response characterized by rapid extravasation of leucocytes and release of excessive amounts of cytokines. We have utilized an in-vitro adhesion assay to understand the molecular mechanisms responsible for SEA-induced extravasation of leucocytes. Stimulation of human umbilical cord endothelial cells (HUVEC) with increasing concentrations of recombinant SEA (rSEA) did not influence the in-vitro adhesion of HL-60 cells to HUVEC, whereas stimulation of HUVEC by interleukin (IL)-1beta supported adhesion of HL-60 cells. Increased adhesion of HL-60 cells to HUVEC was noted upon stimulation of endothelium with culture medium obtained from human peripheral blood mononuclear cells (PBM) stimulated with recombinant SEA for 24 (CM-SEA 24 h), 72 (CM-SEA 72 h) and 120 h (CM-SEA 120 h), but not after stimulation with culture medium obtained from control human peripheral blood mononuclear cells (CM), suggesting that soluble factors present in the supernatants play a major role in SEA-induced cell adhesion. While CM-SEA 24 and 72 h induced both a rapid (4 h) and delayed type of adhesion, CM-SEA 120 h only induced a delayed type of adhesion. Stimulation of PBM by SEA resulted in increased levels of IL-1beta, IL-2 and interferon (IFN)-gamma after 24h. Further stimulation for 72-120h resulted in a significant increase in the levels of IL-1beta, IFN-gamma and tumour necrosis factor (TNF). Stimulation of PBM with SEA also resulted in increased levels of soluble and L-selectin in the cell supernatants. Increased cell-surface expression of E-selectin, ICAM-1, HLA-DR and VCAM-1 was detected on HUVEC stimulated with CM-SEA media. While E-selectin and VCAM were induced on HUVEC within a few hours, induction of ICAM and HLA-DR required a longer induction period. Adhesion of HL-60 cells to HUVEC treated with CM-SEA was inhibited by monoclonal antibodies (MoAbs) against both the selectin and integrin families of cell adhesion molecules, suggesting that multiple pathways contribute to SEA-induced leucocyte extravasation. The results suggested that selectin-dependent adhesion was more prominent during the early phase while integrin-induced adhesion occurred at a later stage.

Effect of anti-apoptotic genes and peptide inhibitors on cytoplasmic acidification during apoptosis.

Cytoplasmic acidification has been shown to occur during the apoptotic process of cell death although its relation with other events in the process are not yet clear. AK-5 tumor cells have been shown to undergo apoptosis upon treatment with stimuli like dexamethasone (1 microM) or with serum from animals that reject AK-5 tumor. The current study was designed to measure the extent of cytoplasmic acidification during apoptosis in AK-5 cells and to study the effect of antiapopoptic genes and peptide inhibitors on cytoplasmic acidification. Our results show that AK-5 cells when triggered into apoptosis show intracellular acidification by about 0.2 pH units and this is prevented when cells are treated with peptide inhibitors. In addition cytoplasmic acidification does not occur when AK-5 cells are transfected with anti-apoptotic genes Nedd-2 A.S, Crm A or bcl-2 which inhibit apoptosis.

Modulation of P-selectin expression on isolated human platelets by an NO donor assessed by a novel ELISA application.

Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method. flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4 degrees C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 x 10(7)/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1-1.0 U/ml), ADP (10 microM) and epinephrine (100 microM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 microM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.

Identification and characterization of promoter elements responsible for the induction of the albumin gene by heat shock in early embryonic rat liver.

We had reported earlier that the expression of albumin increases upon heat shock in embryonic rat liver cells at about 12-13 days of gestation. Here, we report on the identification of heat shock elements (HSEs) within -450 bp of the rat albumin promoter using chloramphenicol acetyl transferase (CAT) assays done with the extracts from H4II-E-C3 cells transfected with plasmids carrying the CAT reporter gene under the control of different deletion fragments of the rat albumin promoter. Gel retardation assays done with synthetic oligonucleotides representing putative HSEs in the rat albumin promoter and H4II-E-C3 cell extracts show that the heat shock factors bind this region in a sequence-specific and reversible manner. Super-shift assays demonstrated that the HSEs present in the rat albumin promoter are bound by HSF1 and not by HSF2. This effect of heat shock on the expression of rat serum albumin is seen only in the liver and is not observed in other tissues, suggesting that HSF-mediated activation of albumin gene cannot overcome the negative regulatory factors present in other tissues. In addition to the HSEs, we have identified a putative GAGA factor binding site in the rat albumin promoter at -228 bp to -252 bp position. These GAGA repeats are bound in a sequence-specific and reversible manner by two factors in a nonstressed cell, whereas only one of these two factors continues to bind the GAGA repeats under heat shock conditions. The physiological significance of these results is discussed.

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.