Crystal structure of the kinesin motor domain reveals a structural similarity to myosin

Kinesin is the founding member of a superfamily of microtubule based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation. It has two identical approximately 960-amino-acid chains containing an amino-terminal globular motor domain, a central alpha-helical region that enables dimer formation through a coiled-coil, and a carboxy-terminal tail domain that binds light chains and possibly an organelle receptor. The kinesin motor domain of approximately 340 amino acids, which can produce movement in vitro, is much smaller than that of myosin (approximately 850 amino acids) and dynein (1,000 amino acids), and is the smallest known molecular motor. Here, we report the crystal structure of the human kinesin motor domain with bound ADP determined to 1.8-A resolution by X-ray crystallography. The motor consists primarily of a single alpha/beta arrowhead-shaped domain with dimensions of 70 x 45 x 45 A. Unexpectedly, it has a striking structural similarity to the core of the catalytic domain of the actin-based motor myosin. Although kinesin and myosin have virtually no amino-acid sequence++ identity, and exhibit distinct enzymatic and motile properties, our results suggest that these two classes of mechanochemical enzymes evolved from a common ancestor and share a similar force-generating strategy.     

Childhood leukemia in Belarus before and after the Chernobyl accident

Childhood leukemia (ICD 204-208 [1]) incidence rates in the different regions of Belarus are reported for a period before and after the Chernobyl accident (1982-1994). There are, at this point, no recognizable trends towards higher rates.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

In vivo purging with high-dose cytarabine followed by high-dose chemoradiotherapy and reinfusion of unpurged bone marrow for adult acute myelogenous leukemia in first complete remission

PURPOSE: To evaluate in a prospective study the efficacy of autologous bone marrow transplantation (BMT) in adult patients with acute myelogenous leukemia (AML) in first remission, using a single course of high-dose Cytarabine (HD Ara-C) consolidation therapy as in vivo purging. PATIENTS AND METHODS: Sixty consecutive adult patients with AML in first complete remission (CR) were treated with HD Ara-C consolidation therapy as a method of in vivo purging before marrow collection. High-dose therapy consisted of fractionated total-body irradiation (FTBI) 12 Gy, intravenous etoposide 60 mg/kg, and cyclophosphamide 75 mg/kg, followed by reinfusion of cryopreserved marrow. RESULTS: Sixty patients underwent consolidation treatment with HD Ara-C with the intent to treat with autologous BMT. Sixteen patients were unable to proceed to autologous BMT (10 patients relapsed, one died of sepsis, one developed cerebellar toxicity, two had inadequate blood counts, and two refused). Forty-four patients underwent autologous BMT and have a median follow-up time of 37 months (range, 14.7 to 68.7) for patients who are alive with no relapse. The cumulative probability of disease-free survival (DFS) at 24 months in the intent-to-treat group is 49% (95% confidence interval [CI], 37% to 62%) and in those who actually underwent autologous BMT is 61% (95% CI, 46% to 74%). The probability of relapse was 44% (95% CI, 31% to 58%) and 33% (95% CI, 20% to 49%) for the intent-to-treat and autologous BMT patients, respectively. CONCLUSION: This approach offers a relatively high DFS rate to adult patients with AML in first CR. The results of this study are similar to those achieved with allogeneic BMT.     

Relation of stressors and depressive symptoms to clinical progression of viral illness

OBJECTIVE: The aim of this research was to determine whether and in whom stressors and depressive symptoms facilitate clinical recurrence of herpes simplex virus (HSV) and progression of HIV. METHOD: Meta-analytic techniques were used to review the relations of stressors and depressive symptoms to clinical recurrence of HSV in 16 published studies and to indicators of HIV progression in 19 published studies. The authors calculated average effect sizes, performed fixed effect and random effect inferential analyses, tested for heterogeneous findings, and identified potential moderating variables. RESULTS: Depressive symptoms were associated with a slightly increased risk of HSV recurrence and increased reports of HIV-related symptoms, whereas stressors were not. However, depressive symptoms were not associated with objective indicators of accelerated HIV progression. Stressor studies, especially those that ascertained population-specific life events, found numerical and functional decrements in circulating natural killer cell populations. The candidate moderators identified include, for HSV recurrence, age, sex, and medication status, and for HIV-related symptoms, age, race, disease stage, and co-infection with HSV. CONCLUSIONS: Depressive symptoms, but not stressors, increase the risk of HSV recurrence generally. Depressive symptoms do not appear to accelerate HIV progression ubiquitously, although they are associated with increased reporting of HIV-related symptoms. Future studies that ascertain population-specific stressors should determine whether reductions in cytotoxic lymphocytes influence HIV disease progression. Moreover, researcher should investigate the role of the identified moderators and recognize psychoimmune moderators in existing and novel study groups. These analyses could confirm that certain individuals are especially susceptible to the effects on disease progression of stressors, depressive symptoms, or both.     

Detection of foodborne pathogens using DNA probes and a dipstick format

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such "gold standard" methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such as Salmonella, Listeria, and E. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.     

Circulating antibodies against p53 protein in patients with ovarian carcinoma. Correlation with clinicopathologic features and survival

BACKGROUND: Genetic alterations of the p53 tumor suppressor protein are the most frequent molecular events in human carcinogenesis. For as yet unknown reasons, mutant p53 often acts as an immunogen for autoantibody generation. These autoantibodies can be detected in the serum of cancer patients. The presence of such antibodies has been identified in a subset of patients with ovarian carcinoma, but their clinical significance has not been investigated. METHODS: Serum samples from patients with ovarian carcinoma were quantitatively analyzed for the presence of p53 autoantibodies with a time-resolved immunofluorometric procedure. Tumor p53 overexpression was assessed by immunohistochemical analysis of tissue sections. Kaplan-Meier survival curves were calculated for p53 antibody positive and negative patients, and the Cox model was used to evaluate the strength of the associations between the presence of serum p53 antibodies and cancer relapse or death, and also between the presence of such antibodies and other clinicopathologic features. RESULTS: p53 antibodies were detected in the serum of 41 of 174 patients with ovarian carcinoma (24%). Antibody levels ranged from a few hundred to 9 x 10(6) arbitrary Units/L, and fluctuated during the course of the disease. p53 antibody positive patients tended to have tumors overexpressing p53, but the association between the two parameters was not statistically significant (P = 0.13). There was also no association between the presence of p53 antibodies and clinical stage, tumor histologic type, or overall patient survival. However, these antibodies were more frequently present in patients older than 50 years (P = 0.001), in patients with moderately or poorly differentiated tumors (P = 0.001), and in patients who received chemotherapy (P = 0.015), and who suffered relapse after surgery (P = 0.018). In univariate analysis, p53 antibody positive patients were at an increased risk for relapse but not death. In multivariate analysis, the differences in disease free and overall survival between patients who were p53 antibody positive or negative were not statistically significant. CONCLUSIONS: p53 autoantibodies are found frequently in the serum of patients with ovarian carcinoma. The presence of such autoantibodies was associated with older patient age, more aggressive tumors, and reduced patient disease free survival. In multivariate analysis the prognostic value of p53 autoantibodies was not statistically significant.     

IgD-receptor up-regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination

Aged individuals (more than 65 years) were classified as antibody (Ab) responders on the basis that they showed increases to more than or = 1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre-immunization samples of blood taken from seven Ab-responders and seven Ab-nonresponders were examined for their ability to exhibit up-regulation of IgD-receptor (IgD-R) after exposure for 2 h to immobilized cross-linked IgD, as shown by rosetting with IgD-coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD-R up-regulation was greater than 5% in all Ab-responders and less than 4% in all the Ab-nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD-R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894. Injection of influenza vaccine itself also induced IgD-R on PBMC in vivo. The percentage of IgD-R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre-injection levels by day 28 in young subjects and aged Ab-responders, but not in Ab-nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab-responders exhibited IgD-R upon immunization in vivo. These findings suggest that Ag injection causes rapid up-regulation of IgD-R by cross-linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD-R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab-responders than in Ab-nonresponders.     

Primary binding domain of bovine von Willebrand factor fragment expressed in E. coli

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.     

Costs and charges associated with three alternative techniques of hysterectomy

BACKGROUND: Many hysterectomies are now performed by a laparoscopically assisted vaginal technique. This procedure is controversial, partly because of concern about cost. We studied hospital charges and costs for the procedure as compared with those for total abdominal hysterectomy and total vaginal hysterectomy in clinically similar groups of patients. METHODS: From hospital-discharge data and patients' charts, we identified hysterectomies performed in 1993 and 1994 by 96 surgeons at a community teaching hospital to treat benign conditions. The patients were grouped according to the surgical procedures performed in conjunction with the hysterectomy. Data on hospital charges and cost-to-charge ratios for 64 hospital cost centers were used to assess charges and costs for specific resources, as well as for the hospitalization overall. RESULTS: Of 1049 patients studied, 26 percent underwent laparoscopically assisted vaginal hysterectomy, 54 percent underwent abdominal hysterectomy, and 20 percent underwent vaginal hysterectomy. The average hospital stays were 2.6, 3.9, and 2.9 days, respectively, and the mean total charges (facility charges plus professional fees) for the hospitalizations were $6,116, $5,084, and $4,221 (P<0.001 for the comparison of the laparoscopic technique with both other techniques). The mean facility costs were $4,914, $3,954, and $3,116, respectively (P<0.001 for the same comparison), with similar findings in all subgroups. The higher charges and costs for laparoscopically assisted vaginal hysterectomy were due to higher supply costs, particularly when disposable supplies were used, and to longer operating-room time. CONCLUSIONS: Despite shorter hospital stays, in-hospital charges and costs for laparoscopically assisted vaginal hysterectomy are higher than for either alternative procedure, because of the disposable supplies that are typically used and the longer operating-room time.