Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity

A new image analysis-based technique was used to quantitatively examine the effects of the "Ca2+-jump" activation protocol on the maintenance of fiber quality in skinned rabbit psoas muscle fiber segments. Specifically, contractions in pCa 4.6 were preceded by short-duration "preactivation" soaks in a solution in which EGTA was replaced with the low-Ca2+ buffering capacity analog hexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca2+ equilibration within the fiber segments. Fiber quality was assessed by examining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 micrometer, thus allowing the majority of the sarcomeres to be visualized in the field of view (x200 and x400 magnification). The preactivation protocol resulted in less deterioration of fiber quality with repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.     

Preoperative anxiety and fear: a comparison of assessments by patients and anesthesia and surgery residents

We sought to compare self-assessment of preoperative anxiety levels and selection of worst fears by surgical patients with the assessments made by the anesthesia and surgery residents providing intraoperative care for those patients. One hundred inpatients at a Veterans Affairs hospital (Group 1) and 45 patients at a University hospital (Group 2) were asked to complete a brief questionnaire; the residents were asked to complete the same questionnaire. Group 1 results showed that median patient visual analog scale (VAS) scores were lower for anxiety about anesthesia compared to surgery (16 vs 22, P < or = 0.05). Anesthesia resident VAS scores were higher than patient or surgery resident scores. Neither type of resident was able to predict their individual patient's VAS score (Kendall's tau). The fear chosen with the greatest incidence by Group 1 patients and residents was "whether surgery would work". A significant number of residents (34%, anesthesia or surgery, P < or = 0.05) matched their patient's fear choice. Residents commonly chose fears related to their specialty (e.g., anesthesia residents chose anesthesia-related fears more often than surgery residents, 50% vs 28%, P < or = 0.001). In Group 2, residents demonstrated an improved ability to predict patient scores. For instance, both surgery and anesthesia residents were able to predict individual University patient VAS scores (P < or = 0.01). The fear chosen with the greatest frequency by Group 2 patients was "pain after the operation". Sixty percent of anesthesia residents matched their patients' fear choice (P < or = 0.001). This study indicates a variable ability of anesthesia and surgery residents to predict patient anxiety and fear which may be due, in part, to difficulty in understanding a Veterans Affairs hospital patient population.     

The lateral retromalleolar groove: a radio-anatomic study]

The anatomy of the malleolar peroneal groove is presented. The results are based on a coupled osteological and CTscan study of 20 samples of fibulae. The average distal fibular torsion was 64 degrees. The peroneal groove was oriented posteriorly (mean value: 78 degrees). Three types of morphological variations were found: concave, flat, convex (the convex shaped groove was the most frequent one: 70%). The average width of the groove was 9 mm. These morphometric results were compared to "clinical" ones performed on patients with a peroneal tendons dislocation syndrome: CTscan study showed an osseous dysplasia concerning the groove depth (flat or convex) and/or a torsional insufficiency. Hypothesis of a bone dysplasia in peroneal dislocation syndrome is discussed.     

Handedness in native Amazonians

Using selected incubation conditions we have identified intermediate steps, between the first glucose transferred to protein and the appropriate substrate for glycogen synthase. Mn2+ stimulates the addition of the first, and probably, the second glucose molecule to the acceptor protein but inhibits further elongation. In the presence of Mn2+ only one radioglucosylated protein band of M(r) 42 kDa was evident. In the absence of Mn2+, two bands of 60.7 and 64.6 kDa were obtained indicating elongation of the glucan chains. After Glc6P addition a family of glucosylated proteins with higher M(r) was obtained, as reported previously. Mn2+ inhibition of the second step, is reversed by PMSF+Glc6P addition. Under these conditions a family of radioglucosylated protein bands with M(r) far in excess of 42 kDa, similar to that obtained without Mn2+, was obtained. Therefore, two different transglucosylating activities were necessary, at least, to prepare the appropriate substrate for glycogen synthase. Based on these observations the model we proposed earlier for glycogen biogenesis is modified. The original "Glycogen Initiator" implies at present two enzymatic activities, Glycogen Initiator 1 (activated by Mn2+) and Glycogen Initiator 2 (inhibited by Mn2+).     

Analysis of patients referred to the gastroenterology service of a VA medical center

We recorded clinical information over a 12-month period on consecutive consultations to the gastroenterology service of the Durham VA Medical Center. Of 902 consultations, 789 were prospectively collected. Eighty-five percent of the patients were between 40 and 70 years old. Seventy-five percent of the referrals were from the internal medicine service. The most frequent reasons for consultation were abdominal pain (19%), GI bleeding (active, 16%; occult, 9%), abnormal results of liver tests (18%), and request for a procedure (11%). Diseases of the liver (32%) and "peptic diseases" (30%) were the most common diagnoses. One or more procedures were done in 71% of consultations. When these data are compared with those of a practicing gastroenterologist, using an identical instrument, it is apparent that trainees' experience with structurally identifiable gastroenterologic disease and with a variety of procedures was similar in scope. There were, however, differences in that the physicians at the VA saw substantially fewer patients with so-called "functional" illness. If these data are applicable to other VA Medical Centers, then the training of physicians in gastroenterology at a VA Medical Center should probably be broadened.     

Sample suitability for the detection of minor white cell populations (microchimerism) by polymerase chain reaction

BACKGROUND: There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing. STUDY DESIGN AND METHODS: Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood. RESULTS: Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed. CONCLUSION: Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.     

Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop

High doses of morphine produce a state of behavioural inactivity and muscular rigidity. This type of 'catalepsy' is clearly different from the state which is produced by the administration of neuroleptics, e.g. haloperidol. While haloperidol-induced catalepsy can easily be antagonised by NMDA receptor antagonists, there has been a report that the non-competitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801) potentiates morphine-induced catalepsy. The aim of this study was to further examine the role of glutamate receptors in the mediation of morphine-induced catalepsy. To this end we coadministered morphine (20, 40, 60 mg/kg i.p.) with MK-801 (0.1 and 0.3 mg/kg i.p.), the competitive NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 37849) (2 and 6 mg/kg i.p.), or 1-(4-aminophenyl)-4-methyl-7,8-methylen-dioxy-5H-2,3- benzodiazepine (GYKI 52466) (2 and 4 mg/kg), an antagonist of the AMPA type of glutamate receptors, respectively. The degree of catalepsy was assessed using two different methods, the 'bar/podium/grid' test which is commonly used to measure neuroleptic-induced catalepsy, and a test for the presence or absence of righting reflexes after turning the animals into a supine position. It was found that in the 'bar/podium/grid' test coadministration of both NMDA receptor antagonists significantly and dose-dependently augmented morphine-induced catalepsy. The results using the AMPA receptor antagonist were less clear since the lower dose of GYKI 52466 tended to attenuate the morphine effect whereas the higher dose augmented morphine-induced catalepsy in some cases. While placing the animals on the bar and on the podium produced essentially the same results, the grid was found to be inapplicable for the measurement of morphine-induced catalepsy since the animals did not cling to the grid and fell off almost immediately after being released from the experimenter's hand. With respect to the righting reflexes it was found that the number of animals not showing these responses increased when MK-801 or CGP 37849 was coadministered with morphine. In contrast, most of the animals treated with GYKI 52466 and morphine displayed intact righting reflexes. It is concluded that glutamatergic transmission plays an important role in the mediation of morphine-induced catalepsy, though different to that of haloperidol-induced catalepsy, and that NMDA and AMPA receptors are differentially involved in different aspects of the associated behavioural state.