Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::beta-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/ antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels

BACKGROUND: The authors previously reported a statistically significant effect of psychosocial intervention on survival time of women with metastatic breast carcinoma. In this study, the authors investigated whether this effect could be explained by differences in the medical treatment patients received subsequent to their group participation or differences in causes of death. METHODS: Of the original 86 study participants, medical treatment charts for 61 and death certificates for 83 were available for further analysis. The authors reviewed the course of the medical treatment they received subsequent to their entry into the randomized psychotherapy trial. RESULTS: Although there were no statistically significant differences with regard to chemotherapy and hormone therapy between the control and treatment groups, women in the control group tended to have received more adrenalectomies, although this procedure did not account for the difference in survival time between the control group and the treatment group. Furthermore, women in the control group developed more bone and lung metastases than the women in the treatment group. CONCLUSIONS: Differences in disease course between the control and treatment groups appeared to be independent of any differences in medical treatment received.     

Parameters affecting the efficiency of affinity-based reversed micellar extraction and separation (ARMES) in glycoprotein purification

The Bladder Neck Support Prosthesis (BNSP) was used in 21 women with combined genuine stress incontinence (GSI) and detrusor instability (DI). Outcomes included frequency volume charts, pad tests, voiding cystometry and quality of life scores, up to the sixth month. Of the 21 recruits, 5 never wore the BNSP home, leaving 16 participants. A further 2 did not reach week 4 because of poor efficacy or inability to fit the device. In the 14 who reached week 4, the median number of leaks/day declined from 4.3 to 1.0 (p = 0.002). Median pad test loss fell from 53 to 7 mL (p = 0.012). Cystometry showed an increase in maximum bladder capacity (p < 0.05) and a modest reduction in severity of detrusor instability, with no evidence of outflow obstruction. Three further women discontinued because of poor efficacy (2) or a poorly fitting device (1), leaving 11 of 16 participants (69%) at week 8, when median pad test loss fell to 2 mL. The BNSP is a useful option in patients with the unfortunate combination of an unstable bladder and an incompetent urethra, but requires careful fitting and attention to detail.     

Vaccination for experimental gliomas using GM-CSF-transduced glioma cells

Within the pituitary, folliculostellate (FS) cells are considered to regulate the intercellular communication between endocrine cells by paracrine mechanisms. One of the possible paracrine factors involved, could be interleukin-6 (IL-6) which is produced by these cells. Since IL-6 has been shown to be a growth factor of pituitary cells, we have determined whether IL-6 can also influence FS cell proliferation. To test this, a FS cell-like mouse pituitary cell line (TtT/GF cells) was used that exhibits most characteristics of normal FS cells. Under serum-free conditions the proliferation of TtT/GF cells is critically dependent on the initial seeding density: cells seeded at low density do not grow at all whereas cells seeded at high density proliferate with maximal doubling times of 34 hrs. Mouse IL-6 (mIL-6) stimulated only low density cell cultures in a dose and time dependent manner. For cells seeded at high density, exogenously added IL-6 may have failed to stimulate growth because of endogenously produced mIL-6. Conditioned medium from TtT/GF cells, in which mIL-6 concentrations up to 1017 pg/ml were measured, stimulated the proliferation of TtT/GF cells, indicating an autocrine growth stimulatory mechanism. A neutralizing mIL-6 antibody partially suppressed the growth of TtT/GF cells seeded at high density and partially reduced the growth stimulatory activity of TtT/GF conditioned medium. Thus, IL-6 is one but not the only factor that is involved in the autocrine growth stimulatory loop. The relevance of this mechanism for normal FS cells and its physiological consequence needs to be elucidated.     

Long-term outcome in acute myocardial infarction patients admitted to hospitals with and without on-site cardiac catheterization facilities. MITI Investigators. Myocardial Infarction Triage and Intervention

BACKGROUND: Previous studies have documented the strong association between availability of on-site cardiac catheterization facilities and increased use of coronary angiography in patients with acute myocardial infarction (AMI). Although these studies have shown little influence of the availability of catheterization labs on hospital mortality, no long-term follow-up has been reported. METHODS AND RESULTS: From a cohort of 12,331 AMI patients admitted to 19 Seattle area hospitals, we compared long-term outcome in 7985 patients admitted to hospitals with and 4346 patients admitted to hospitals without on-site catheterization labs. During the index hospitalization, patients admitted to hospitals with on-site catheterization were more likely to undergo coronary angiography (67.1% versus 39.3%, P<.0001), coronary angioplasty (32.5% versus 13.2%, P<.0001), or coronary bypass surgery (12.5% versus 9.5%, P<.0001). At 3-year follow-up, patients admitted to hospitals with on-site catheterization labs were more likely to undergo postdischarge angiography (19.2% versus 15.2%, P=.0001) and coronary angioplasty (11.6% versus 8.2%, P<.0001). This was associated with approximately $2500.00 per patient in higher cumulative costs. Despite this higher rate of procedure use, there was no association between admission to a hospital with on-site catheterization facilities and lower long-term mortality (multivariate hazard ratio, 1.0; 95% CI, 0.93 to 1.1., the hazard being associated with admission to hospitals with on-site catheterization facilities). CONCLUSIONS: In an urban area with unconstrained patient transfer mechanisms and high overall cardiac procedure use rates, AMI patients admitted to hospitals without on-site catheterization facilities were managed with fewer procedures during hospitalization and follow-up. This more conservative treatment approach was not associated with any observed increase in long-term mortality.     

The Women's Health Curriculum by a problem-based learning method for medical students at the University of California, San Francisco

OBJECTIVES: Our objectives were to (1) expand and strengthen the women's health curriculum at the University of California, San Francisco, and (2) evaluate the responses of both medical students and faculty to this curriculum. STUDY DESIGN: A written evaluation of the curriculum in women's health was completed by both students and faculty. Variables studied included mean scores of cases, the overall course score, and the preferences of medical students for faculty specialty in teaching the small groups. RESULTS: The overall course evaluation score was 7.81 (range 1 to 10). For those students who had both faculty from internal medicine or family medicine and obstetrics and gynecology, there was a strong preference that obstetrician-gynecologists teach the majority of the cases. CONCLUSIONS: The new case-based curriculum in women's health was enthusiastically received by both medical students and faculty.