Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.

The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of Mr = 95,000 and 35,000 +/- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (Mr = 95,000) and proteinase II (Mr = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

Sexual dimorphism of blood pressure: Possible role of the renin-angiotensin system

The prevalence of hypertension in men is higher than in women and the onset of this disease is earlier in male than in female subjects. In spontaneously hypertensive rats, males also have higher blood pressures than females. Evidence from epidemiological, physiological, molecular biological and morphological studies concerning this sexual dimorphism is reviewed. We demonstrate that the gonadal steroids testosterone and estrogen have important effects on the gene regulation of the renin-angiotensin system. This may in part contribute to the sexual dimorphism in blood pressure control. The direct effect of steroid hormones on genes related to hypertension provides a suitable paradigm to improve our understanding of molecular and cellular mechanisms of cardiovascular control.     

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.     

Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([³H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-γ (IFN-γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-γ's ability to control cell growth and induce an antiviral state.     

A flow cytometric study of chromosomes from rat kangaroo and chinese hamster cells

Summary Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was optimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10 DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated into 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in the karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing high power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influenced by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.