Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus

A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Accumulation of the labdane diterpene Marrubiin in glandular trichome cells along the ontogeny of Marrubium vulgare plants

The content of the diterpene Marrubiin was assessed by GC-FID in leaves of Marrubium vulgare plants along their ontogeny. Maximum accumulation occurred just before flowering time and in fully expanded leaves. After feeding the plants with radio labeled [³H]-geranyl geranyl diphosphate, up to 70% of the radioactivity was recovered in HPLC-Rt coincidental with authentic Marrubiin, which was also characterized by GC-EIMS, thus confirming that the biosynthesis of Marrubiin proceeds through the 1-deoxy-D-xylulose-5-phosphate pathway. The major accumulation of radioactivity occurred in glandular trichome cells, and the product remained stable throughout.     

The metalloproteinase ADAM‐12 regulates bronchial epithelial cell proliferation and apoptosis

Abstract. Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.     

Mitro-aortic pathology: a point of view for a fully transcatheter staged approach

Severe aortic valve stenosis (AVS) and mitral valve regurgitation (MVR) often coexist. Although a fully percutaneous treatment for the two conditions, by means of transcatheter aortic valve implantation (TAVI) followed by MitraClip, can be appealing in selected high-risk candidates, critical and strategical reasoning should be applied. In a 3-year period we have developed a single-centre experience of 14 patients who were managed with a staged percutaneous approach to treat severe AVS and MVR. The average interval from TAVI to MitraClip repair was 101 ± 12 days. Success for TAVI was 100% and 92.9% (13/14) for MitraClip. At late follow-up, 3 patients developed MVR 3+. Estimated 1?year survival was 66.5%. Freedom from 1?year endpoint (death, stroke, major bleeding, myocardial infarction, and cardiac re-hospitalisation) was 57.9%.In our view, a fully transcatheter approach for mitro-aortic pathology is feasible and should be performed only as a staged procedure in those patients that remain symptomatic, in spite of successful TAVI. It should be emphasised that although the periprocedural success rate is satisfactory, follow-up mortality and re-hospitalisation rates remain high, even at mid-term follow-up. This most probably results from the advanced clinical picture at time of referral for treatment.     

Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids

Hep G2, a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents, has been found to express a wide variety of liver-specific metabolic functions. Among these functions are those related to cholesterol and triglyceride metabolism. Confluent Hep G2 monolayers express normal low-density lipoprotein (LDL) receptors and continue to internalize and metabolize chylomicrons, very low-density lipoproteins (VLDL), LDL, and high-density lipoproteins. In lipoprotein-free medium, apolipoproteins A-I, A-II, B, C, and E accumulate in the medium together with cholesterol, cholesteryl ester, triglyceride, and all the primary bile acids. The regulation of their synthesis and secretion is not fully known and their interrelationships have not been established. Because Hep G2 cells express these and other components of cholesterol and triglyceride metabolism, they are a microcosm for studying the central role of the liver.     

Effects of opioid blockade with nalmefene in older impotent men

We evaluated the effect of the opioid antagonist nalmefene on the HPG axis and on food consumption in 14 older impotent men. These patients had low to low normal mean serum testosterone values and normal gonadotrophin levels on screening evaluation. Normal response to GnRH was demonstrated in all the men. The protocol called for 24 hours of evaluation before and during administration of nalmefene 2.0 mg IV every 8 hours for 3 doses. During each 24 hour period, the following determinations were made: serum testosterone, FSH, and LH by five separate determinations between 8 AM and noon; 8 AM and 11 PM serum cortisols; 24 hour urine collections for free cortisol; and nocturnal penile tumescence (NPT). Food consumption was measured from 4 PM to 10 AM during the two periods. Nalmefene resulted in significant rises in testosterone, LH, and FSH. Nalmefene significantly elevated morning and evening cortisol measurements in all the patients. Nalmefene decreased total calorie consumption, principally by decreasing fat consumption. There was no effect on NPT. We conclude that in older impotent men, nalmefene acutely increases activity of the HPG axis and decreases calorie intake predominantly by decreasing fat consumption.     

A Neuro-Comparative Study between Single/Successive Thorium Dose Intoxication and Alginate Treatment

The adult male albino rats were grouped into five groups (control group and four variably treated groups with thorium (Th) in single or successive with or without alginate treatment). The IP administration of thorium nitrate (13.6 mg/kg b.wt.) induced a regional distribution and accumulation ordered as cerebellum > cerebral cortex > brain stem > hippocampus > hypothalamus > striatum. Also, it induces a significant increase in Na⁺, Ca²⁺, and Fe³⁺ ion content and malondialdehyde (MDA) level while K⁺ ions and glutathione (GSH) level were significantly decreased. On the other hand, the daily oral administration of 5% alginate showed a significant decreasing in the accumulation of thorium in the different brain areas and mitigated its hazardous effects. By the alginate treatment, Na⁺, Ca²⁺, Fe³⁺, and level of MDA were declined while K⁺ ions and GSH level showed a significant increase. The improvement of the investigated parameters was attributed to the specific chelating, regeneration, and antioxidant properties of the alginate. So, alginate administration could ameliorate the hazardous effects of thorium nitrate.