LCMS Determination and Cytotoxicity of Abrus precatorius on L6 and SK-N-MC Cell Lines.

OBJECTIVE: The present study aimed to investigate the cytotoxic effect of various extracts derived from Abrus precatorius Linn. leaves on rat L6 and human SK-N-MC neuroblastoma cell lines and determine the secondary metabolites responsible for the cytotoxicity of Abrus precatorius. METHODS: Successive solvent extraction of A. precatorius leaves was carried out using the Soxhlet apparatus with solvents such as petroleum ether, chloroform, ethyl acetate, and ethanol. HPTLC fingerprinting and LC-MS studies were performed to assess the presence of secondary metabolites, such as flavonoids and phenols, in the ethyl acetate extract. Furthermore, the cytotoxic effect of extracts was tested on rat skeletal muscle cell line L6 and human neuroblastoma cell line SK-N-MC using MTT assay. RESULTS: The total phenolic content of ethyl acetate and ethanol extracts of A. precatorius were 72.67 and 60.73 mg, respectively, of GAE/g dry weight of the extract. The total flavonoid content of ethyl acetate and ethanol extract of A. precatorius were 107.33 and 40.66 mg of Quercetin equivalents/g dry weight of the extract. LCMS analysis demonstrated that the flavonoids in specific Naringenin, Diosmetin, Glycitin, and Genistein might play a prominent role in the cytotoxicity of A. precatorius. The cytotoxicity study revealed that the extracts of A. precatorius were non-toxic to rat L6 myotubes, and the IC50 values of the various extracts, such as APPE, APCH, APEA, and APET, were >100 µg/ml. The extracts exhibited cytotoxic activity against human neuroblastoma SK-N-MC cells, and the IC50 values of APPE, APCH, APEA, APET, and the standard drug "Cisplatin" were >100, >100, 64.88, >100, and 3.72 µg/ml, respectively. CONCLUSION: It was concluded from the study that the extracts of Abrus precatorius were cytotoxic to neuroblastoma cell lines but non-toxic to normal cell lines. HPTLC and LC-MS studies confirmed that flavonoids in the ethyl acetate extract could be responsible for the biological activity.

Increased incidence of migraine in women correlates with obstetrics and gynaecological surgical procedures.

Migraine is a common chronic neurological disorder; yet no possible aetiology has been identified so far. There is a debate that migraine worsens in women who undergo procedures such as hysterectomy, dilation and curettage (D&C) or cesarean section for delivery. Hence, the present study was attempted to explore the link between procedures like D&C, hysterectomy and cesarean section for delivery and the prevalence of migraine in women. A total of 185 migraine patients were screened based on the inclusion and exclusion criteria of the International Headache Classification guidelines and 70 females who satisfied the inclusion criteria were included for the study. Of the 70 female patients, the numbers of married and unmarried women were 47 and 27, respectively. About 36 married women (80%, 95% CI: 0.146-0.104) had undergone the procedures related to obstetrics and gynaecology as per their medical history. Interestingly, 12 patients (33%, 95% CI: 0.148-0.176) had not experienced migraine attack prior to the above mentioned surgeries. Although, the age adjusted incidence of diagnosed migraine per 100,000 populations showed higher risk between 16-20 years of age (95% CI: 0.104-0.121), significant risk (95% CI: 0.086-0.113) was also observed in the women of 31-35 years age group in the present study. Based on the present study, surgeries such as D&C, hysterectomy and cesarean section for delivery increased the prevalence of migraine in women. Therefore, such procedures should be avoided unless otherwise essential, particularly in patients with positive past history of migraine.

Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells.

Members of the tetraspanin superfamily of proteins are implicated in a variety of complex cell processes including cell fusion. However, the contribution of individual tetraspanins to these processes has proved difficult to define. Here we report the use of recombinant extracellular regions of tetraspanins to investigate the role of specific members of this family in the fusion of monocytes to form multinucleated giant cells (MGC). In contrast to their positive requirement in sperm-egg fusion, previous studies using antibodies and knockout mice have indicated a negative regulatory role for tetraspanins CD9 and CD81 in this process. In an in vitro model of fusion using human monocytes, we have confirmed observations that antibodies to CD9 and CD81 enhance MGC formation; however, in contrast to previous investigations, we found that all members of a panel of antibodies to CD63 inhibited fusion. Moreover, recombinant proteins corresponding to the large extracellular domains (EC2s) of CD63 and CD9 inhibited MGC formation, whereas the EC2s of CD81 and CD151 had no effect. The potent inhibition of fusion and binding of labelled CD63 EC2 to monocytes under fusogenic conditions suggest a direct interaction with a membrane component required for fusion. Our findings indicate that the tetraspanins CD9, CD63 and CD81 are all involved in MGC formation, but play distinct roles.

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.