Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract on U937 and K562 cells.

The antiproliferative, cytotoxic and apoptogenic activities of Bufo melanostictus (Indian common toad) skin extract (TSE) on U937 and K562 leukemic cell line has been investigated. TSE significantly (P<0.001) reduced the time-dependent cell proliferation and decreased MTT values in U937 and K562 cells. TSE (IC50 doses) suppressed the proliferating cell nuclear antigen expression in both the cells. It was demonstrated that, TSE (IC50 doses) primarily arrested the U937 and K562 cells at G1 phase of the cell cycle. Confocal microscopy showed the altered fragmented nuclei and apoptotic bodies formation in TSE (IC50 doses) treated U937 and K562 cells. Membrane blebbing, cell surface shrinkage and perforation were observed through scanning electron microscope. TSE-induced DNA fragmentation in U937 and K562 cells was reflected in single-cell gel electrophoresis. TSE significantly (P<0.001) increase the length-width ratio of DNA mass as compared to control in comet assay. The flow cytometric analysis of annexin-V binding to the cancer cells further supported the apoptotogenic activity of TSE. The effect of TSE on normal human peripheral blood mononuclear cells viability and cytotoxicity was studied in culture and found to be less cytotoxic than on the U937 and K562 cells. The findings from the present study suggested that TSE might possess potent antineoplastic agent having antiproliferative, cytotoxic and apoptogenic activity against U937 and K562 myeloid leukemic cells.     

Competitive binding to a charged leucine motif represses transformation by a papillomavirus E6 oncoprotein

E6 oncoproteins from HPV-16 and bovine papillomavirus type 1 (BPV-1) bind to similar leucine-rich peptides termed charged leucine motifs found on the cellular focal adhesion protein paxillin and the E3 ubiquitin ligase E6AP. BPV-1 E6 (BE6) mutants that do not bind to paxillin are defective at inducing cellular transformation. It is possible, however, that BE6 mutants that do not bind paxillin are defective for transformation for an unrelated reason than the ability to bind to charged leucine motifs. To address the role of BE6 interaction with charged leucine motifs, we fused a BE6-binding charged leucine motif to the amino terminus of BE6, thereby creating an autoinhibitory binding domain. We found that the fusion protein failed to bind to paxillin or transform murine C127 cells. Mutation of the amino terminal binding motif in the fusion protein restored both interaction with paxillin and transformation. This demonstrates that BE6 transformation requires binding to charged leucine motifs on particular cellular proteins and that transformation by papillomavirus oncoproteins can be repressed by competitive interactions with charged leucine motifs.     

Cystic duct and Heister's "valves"

The anatomy and physiology of the cystic duct have been relatively neglected by anatomists and the function of the spiral mucosal folds or "valves" of Heister, first described in 1732, remains obscure. The gross and microscopic anatomy of the cystic duct is reviewed together with results from laboratory investigations into the function of the cystic duct and its spirally arranged folds. The duct and spiral folds contain muscle fibers responsive to pharmacologic, hormonal, and neural stimuli. There is, however, no convincing evidence of a discrete muscular sphincter within the duct. Although the cystic duct is unlikely to play a major role in gallbladder filling and emptying, it appears to function as more than a passive conduit. Coordinated, graded muscular activity in the cystic duct in response to hormonal and neural stimuli may facilitate gallbladder emptying. The principal function of the internal spiral folds that are found in man and other animals may be to preserve patency of this narrow, tortuous tube rather than to regulate bile flow.     

Immunomodulation of autoimmune and inflammatory diseases with intravenous immunoglobulin

Abstract Intravenous immunoglobulin (IVIg) has been used in the treatment of primary and secondary antibody deficiencies for over two decades. Since the early 1980s, the therapeutic efficacy of IVIg has been established in idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, dermatomyositis and Kawasaki syndrome, and the prevention of graft versus host disease in recipients of allogeneic bone marrow transplants. Its use has also been reported in a large number of other autoimmune and systemic inflammatory conditions. In this review, we discuss the mechanisms by which IVIg exerts immunomodulatory effects in immune pathologies.     

Use of alkaline phosphatase to correct the underestimation of fosphenytoin concentration in serum measured by phenytoin immunoassays

The pharmacologic activity of fosphenytoin, a new phosphate ester pro-drug of phenytoin, is due to in vivo conversion to phenytoin. Fosphenytoin concentrations cannot be accurately estimated by phenytoin immunoassays (fluorescence polarization and chemiluminescence) owing to the nonlinear relation between fosphenytoin concentration and the observed cross-reactivity. The problem of slow conversion of fosphenytoin to phenytoin in serum in vitro can be circumvented by rapidly converting fosphenytoin to phenytoin in vitro by alkaline phosphatase. Drug-free serum, heparin, EDTA, or citrated plasma were supplemented with 2 concentrations of fosphenytoin. Then to 1-mL aliquots of specimen, no enzyme (control), 10 microL, or 25 microL of enzyme solution was added. The specimens were incubated, and phenytoin concentrations were measured by fluorescence polarization and chemiluminescent assays. In the absence of enzyme, we observed little conversion of fosphenytoin to phenytoin, but in the presence of only 10 microL of enzyme, the conversion of fosphenytoin to phenytoin was complete in 5 minutes. We also observed complete conversion of fosphenytoin to phenytoin by alkaline phosphatase in heparin, EDTA, and citrated plasma. If clinically indicated, the phenytoin concentration can be measured before and after addition of enzyme to roughly estimate the rate of conversion.     

Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction

Botulinum neurotoxin (BoTx) serotype E blocks spontaneous and evoked quantal release of acetylcholine at the rat neuromuscular junction. Increasing extracellular Ca2+ to 8 mmol l-1 or substituting Ca2+ with La3+ (0.1 and 1.0 mmol l-1) or depolarizing the nerve terminals by 20 mmol l-1 K+ markedly increases miniature end-plate potential frequency in normal muscle, but in BoTx-E poisoned preparations none of these ions, with the exception of 1 mmol l-1 La3+, was able to restore spontaneous quantal transmitter release to levels recorded at unpoisoned junctions. In absolute values the enhancement with La3+ was much less than that reported at normal junctions. Nerve stimulation in the presence of 3,4-diaminopyridine (10-20 mumol l-1) and high calcium (8 mmol l-1) evoked multiquantal end-plate potentials and muscle twitches. We conclude that the neuromuscular block produced by BoTx serotype E is similar to that previously described for BoTx serotype A but differs from that produced by BoTx serotypes B, D and F in not causing desynchronization of nerve impulse-evoked transmitter release. 3,4-Diaminopyridine might be useful in the treatment of poisoning by BoTx serotype E since it markedly enhanced synchronous transmitter release from poisoned motor nerve terminals.     

Antitumor effect of i.p. dopamine in mice bearing Ehrlich ascites carcinoma

Summary The cancer chemotherapeutic efficacy of dopamine (DA) was evaluated in female strain A mice bearing transplantable Ehrlich ascites carcinoma. The results demonstrated significant inhibition of tumor growth with appreciable increase in the host survival time following DA treatment. Diminished activity of the growth-related respiratory enzyme succinate dehydrogenase along with stimulated activity of the lysosomal enzyme, -glucuronidase in DA-treated tumor cells indicated inhibition of tumor growth as well as active lysis of the tumor cells. The direct effect of this compound on tumor proliferation was demonstrated by marked inhibition of DNA synthesis. RNA synthesis was only marginally inhibited.Abbreviations DA Dopamine - EAC Ehrlich ascites carcinoma - SDH Succinate dehydrogenase - -Glu -glueuronidase - ILS Increase of life span