Z/I and A-band lattice spacings in frog skeletal muscle: effects of contraction and osmolarity

A-band and Z-line/I-band lattice spacings were measured by small-angle X-ray diffraction from relaxed and isometrically-contracting whole frog sartorius muscles with lattice spacings reduced or swollen by changing the osmolarity of the bathing solution. A-band spacing increased by approximately 3% upon isometric contraction at reduced lattice spacings (245-356 mOsm) and decreased by approximately 1% at swollen spacings (172 mOsm), similarly to the behaviour of skinned muscles upon changing from the relaxed state to rigor. The Z/I lattice underwent a significant lattice expansion (3-8%) upon isometric contraction at all osmolarities, in qualitative agreement (but quantitative disagreement) with results from electron microscopy on mammalian skeletal muscle. Lattice areas calculated for the Z/I and A-band lattices indicate a barrel-shaped sarcomere in the resting state, which may provide a partial explanation for how longitudinal forces produced in the A-band can produce a radial expansive force in the Z-line during contraction. The radial component of cross-bridge stiffness was calculated from the A-band data for contracting muscle, using a lattice stability model incorporating structural, osmotic and electrostatic forces. The calculations gave a radial cross-bridge stiffness during contraction of about 9 x 10(5) N m-2, and outward radial force per thick filament in normal Ringer's solution of 6 x 10(-9) N, corresponding to a radial force per cross-bridge of 10(-11) N.     

The influence of advanced age on the outcome of assisted reproduction

This study investigates within-subject variations and associations of salivary viscosities and flow rates in a test panel of healthy adults. After several practice sessions, unstimulated and stimulated whole saliva samples were collected 5 times daily (at 0800, 1100, 1400, 1700, and 2000 h) from 30 university students. There was a significant within-subject variation in viscosity and flow rate of unstimulated saliva (P<0.001). Intra-item correlations were significantly different for salivary flow rates (r= 0.82 for unstimulated, r= 0.88 for stimulated, P< 0.001) and viscosity of unstimulated saliva (r= 0.54, P< 0.05), but viscosity of stimulated saliva was different in this respect. Our results indicate that there is a significant within-subject variation in viscosity of unstimulated saliva.     

Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the beta1 and the alpha2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.     

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.     

Genetic determinants of p53-induced apoptosis and growth arrest

Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.     

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.     

The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.