The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.     

Localization of Chlamydia trachomatis heat shock proteins 60 and 70 during infection of a human endometrial epithelial cell line in vitro

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.     

Effect of aluminum on cytoplasmic Ca2+ homeostasis in root hairs of Arabidopsis thaliana (L.)

Aluminum inhibition of root growth is a major world agricultural problem where the cause of toxicity has been linked to changes in cellular calcium homeostasis. Therefore, the effect of aluminum ions (Al) on changes in cytoplasmic free calcium concentration ([Ca2+]c) was followed in root hairs of wild-type, Al-sensitive and Al-resistant mutants of Arabidopsis thaliana (L.) Heynh. Generally, Al exposure resulted in prolonged elevations in tip-localized [Ca2+]c in both wild-type and Al-sensitive root hairs. However, these Al-induced increases in [Ca2+]c were not tightly correlated with growth inhibition, occurring up to 15 min after Al had induced growth to stop. Also, in 32% of root hairs examined growth stopped without a detectable change in [Ca2+]c. In contrast, Al-resistant mutants showed little growth inhibition in response to AlCl3 exposure and in no case was a change in [Ca2+]c observed. Of the other externally applied stresses tested (oxidative and mechanical stress), both were found to inhibit root hair growth, but only oxidative stress (H2O2, 10 microM) caused a prolonged rise in [Ca2+]c similar to that induced by Al. Again this increase occurred after growth had been inhibited. The lack of a tight correlation between Al exposure, growth inhibition and altered [Ca2+]c dynamics suggests that although exposure of root hairs to toxic levels of Al causes an alteration in cellular Ca2+ homeostasis, this may not be a required event for Al toxicity. The elevation in [Ca2+]c induced by Al also strongly suggests that the phytotoxic action of Al in root hairs is not through blockage of Ca2(+)-permeable channels required for Ca2+ influx into the cytoplasm.     

Pathologic features from prostate needle biopsy and prognosis after I-125 brachytherapy

To evaluate the role of detailed pathologic features in predicting outcome for early-stage prostate cancer treated with I-125 brachytherapy. The pretreatment biopsy slides of 103 patients with T1/T2 and Gleason scores of 4-7 prostatic carcinoma, which was treated by transperineal I-125 implantation, were reviewed retrospectively by a single pathologist (P.B.G.). Biochemical tumor control rates [prostate-specific antigen (PSA) below 1.0] were correlated with pretreatment PSA, Gleason score, the amount of tumor in the biopsy samples, and the presence of perineural invasion. In Cox proportional-hazard, multivariate analysis, the strongest predictors of failure were pretreatment PSA above 10 ng/ml (P = 0.013) and the length of the biopsy specimen replaced by tumor (P = 0.15). The percent of biopsy tissue replaced by tumor (P = 0. 74), perineural invasion (P = 0.78), and Gleason score (P = 0.66) were less predictive of prognosis. It was concluded that pretreatment PSA is the strongest predictor of biochemical failure. Detailed assessment of pathological features on needle biopsy added little prognostic information beyond that of pretreatment PSA alone. Like all other prognostic parameters for prostate cancer, there is considerable overlap in pathologic features between those patients who will or will not be controlled biochemically.     

Effect of clozapine on motor function in schizophrenic patients

It is well established that clozapine is less likely than typical antipsychotic drugs to cause clinically discernible extrapyramidal side-effects. There is a paucity of data, however, on clozapine's motor effects. In this report we compare normal controls to groups of chronic schizophrenic patients treated with either typical antipsychotic drugs or with clozapine. Motor function was measured with a target-matching task, a test relying on submaximal sustained force control. Results indicated that patients on clozapine performed with significantly lower accuracy (greater variability) of force control. Even though the clozapine patients were treatment resistant to typical antipsychotic drugs, and many had a history of tardive dyskinesia, we postulate that the observed deficit is likely due to clozapine treatment rather than to earlier treatments or other factors. The observed force control deficit may be the result of an increase in myoclonus and a generally lower level of overall motor activity.     

Palliative care in undergraduate medical education. Status report and future directions

OBJECTIVE: To describe the status of palliative care education in the undergraduate medical curriculum and to offer recommendations for improvement. DATA SOURCES: Review of literature on palliative care and of recently submitted grants on medical education for end-of-life care. STUDY SELECTION: English-language reports of educational programs targeted toward medical students were examined, as well as surveys of medical schools. DATA EXTRACTION: Studies were reviewed by the authors to assess the quality of the educational program, evaluation methodology, and conclusions. From over 9000 citations on palliative care and related topics that were retrieved from MEDLINE searches from 1980 through 1995, and from reviewing 14 palliative care journals published from 1985 through 1996, 310 articles were identified that addressed medical education for end-of-life care, and 180 were carefully examined. DATA SYNTHESIS: While nearly all medical schools offer some formal teaching about end-of-life care, there is considerable evidence that current training is inadequate, most strikingly in the clinical years. Teaching about palliative care is received favorably by students, positively influences student attitudes, and enhances communication skills. However, curricular offerings are not well integrated; the major teaching format is the lecture; formal teaching is predominantly preclinical; clinical experiences are mostly elective; there is little attention to home care, hospice, and nursing home care; role models are few; and students are not encouraged to examine their personal reactions to these clinical experiences. CONCLUSIONS: The increasing attention to palliative care education has created major opportunities for improving education about care at the end of life. Educational programs should be rigorously evaluated to identify best educational practices.     

Seasonal variation in laboratory response to behavioral chemicals of the southern pine beetle

The response ofDendroctonus frontalis to an attractant mixture (frontalin,trans-verbenol, and loblolly pine turpentine) was measured in the laboratory over a four-year period. Beetle response was highest in late winter and early spring, and lowest in midsummer and early fall. Males consistently responded higher than females. Female beetles displayed significantly higher responses in early morning and late afternoon than in the middle of the day. Analysis of beetle pronotal width and fat content revealed a high degree of correlation between these two parameters in female beetles, but there was no correlation of response with either fat content or pronotal width for either sex. There was no evident relationship between mean monthly beetle response and total amounts of frontalin andtrans-veibenol found in hindgut extracts. Daily temperature in months both during which beetles were bioassayed and immediately prior to bioassay was highly correlated to response to the attractant.Texas Agricultural Experiment Station Paper Number 16652. This research was supported by McIntire-Stennis Project 1525, and the U.S. Department of Agriculture-sponsored program entitled The Expanded Southern Pine Beetle Research and Applications Program through TAES-SEA-CR Grant 904-15-4. The findings, opinions, and recommendations expressed herein are those of the authors and not necessarily those of the U.S. Department of Agriculture. All programs and information of the Texas Agricultural Experiment Station are available to everyone without regard to race, color, religion, sex, age, or national origin.     

Prognostic value of bacteriological studies in peritonitis

Based on multiple bacteriological assays of lymph and blood, it is concluded that single detection of pathogenic microorganisms in lymph and blood should not be considered an unfavourable prognostic sign. A provisional external drainage of the thoracic duct would reduce intoxication in patients with peritonitis, and it is estimated as a valuable prophylactic method of combating against bacteriemia.     

Gonadoblastoma: clinicopathologic correlation in six patients

Six patients with a total of nine gonadoblastomas are presented; three--and possibly a fourth--had dysgerminomatous overgrowth which was massive in two patients. Calcification detected by abdominal films was present in three sufficient for preoperative diagnosis. All patients were found to have a Y stem line on peripheral leukocyte chromosome cultures except one patient, who had a 46 XX/45 XO karyotype. She was found to have Y chromatin bodies in the germ cells of her tumor which was in a normal ovary found at exploration for an ectopic pregnancy. Three were found in virilized phenotypic females investigated for amenorrhea, and two for therapy of pelvic masses due to dysgerminomatous overgrowth. Y chromatin studies are reported on gonadal tissue.