Hypohydration effects on skeletal muscle performance and metabolism: a 31P-MRS study

The purpose of this study was to determine whether hypohydration reduces skeletal muscle endurance and whether increased H+ and Pi might contribute to performance degradation. Ten physically active volunteers (age 21-40 yr) performed supine single-leg, knee-extension exercise to exhaustion in a 1.5-T whole body magnetic resonance spectroscopy (MRS) system when euhydrated and when hypohydrated (4% body wt). 31P spectra were collected at a rate of one per second at rest, exercise, and recovery, and were grouped and averaged to represent 10-s intervals. The desired hydration level was achieved by having the subjects perform 2-3 h of exercise in a warm room (40 degrees C dry bulb, 20% relative humidity) with or without fluid replacement 3-8 h before the experiment. Time to fatigue was reduced (P < 0.05) by 15% when the subjects were hypohydrated [213 +/- 12 vs. 251 +/- 15 (SE) s]. Muscle strength was generally not affected by hypohydration. Muscle pH and Pi/beta-ATP ratio were similar during exercise and at exhaustion, regardless of hydration state. The time constants for phosphocreatine recovery were also similar between trials. In summary, moderate hypohydration reduces muscle endurance, and neither H+ nor Pi concentration appears to be related to these reductions.     

National survey of blindness and low vision in Lebanon

Here we report a case that presented with sudden onset of neurological symptoms and was treated with ganciclovir. Polymerase chain reaction analysis for human herpes virus-6 (HHV-6) from his cerebrospinal fluid was later found to be positive. He subsequently recovered with minimal residual neurological symptoms. Encephalitis secondary to this virus may be more common than previously thought in patients undergoing bone marrow transplantation. This condition should be considered in the differential diagnosis of sudden onset neurological symptoms after bone marrow transplantation.     

Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.     

Subunit structure and function of porcine factor Xa-activated factor VIII

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.     

Maternal immunization to Gov system alloantigens on human platelets

BACKGROUND: Immunization to platelet alloantigens can occur during pregnancy or after the transfusion of blood components. Platelet alloantibodies can cause neonatal alloimmune thrombocytopenia and posttransfusion purpura. Transfusion-induced alloimmunization to a novel platelet alloantigen system, Gov, expressed on the 175-kDa glycosyl phosphatidylinositol-anchored platelet glycoprotein, CD109, was previously described. This report describes three unrelated patients who were alloimmunized to Gov(a) or Gov(b) during pregnancy. STUDY DESIGN AND METHODS: Platelets were typed by using radioimmunoprecipitation for HPA-1a, -3a, -5a, -5b, Gov(a), and Gov(b) and by polymerase chain reaction-restriction fragment length polymorphism for HPA-1a, -1b, -3a, and -3b. Maternal sera were screened for platelet antibodies by using radioimmunoprecipitation and the antigen capture assay. RESULTS: Patients 1 and 2 were investigated after the diagnosis of neonatal alloimmune thrombocytopenia in their children, and alloantibodies specific for Gov(b) and Gov(a), respectively, were detected in maternal serum. Serum from patient 3, who had mild idiopathic thrombocytopenia purpura with no detectable autoantibody, was found to contain alloantibodies to Gov(b) and to HPA-5b, presumably as a result of immunization during pregnancy. Platelet typings confirmed that the patients were at risk for alloimmunization to the respective antigen. CONCLUSION: This report of three cases of maternal alloimmunization to antigens in the Gov system indicates that immunization can occur via placental transfer of antigen and that Gov system alloantibodies may be associated with neonatal alloimmune thrombocytopenia.     

A region within murine chromosome 7F4, syntenic to the human 11q13 amplicon, is frequently amplified in 4NQO-induced oral cavity tumors

Our previous reports have shown that two thirds of 4-nitroquinoline-1-oxide (4NQO)-induced murine oral squamous cell carcinomas (SCC) have Hras1 mutations. Loss of heterozygosity (LOH) involving the distal portion of chromosome (Chr) 7 occurred in half of the tumors with Hras1 mutations. Here, we demonstrate that five of six tumors with LOH have 4-8-fold amplification involving the distal portion of Chr 7 (7F4). Ccnd1. Fgf4 and Fgf3, within the most telomeric region of Chr 7 (70.5 cM), are co-amplified. The region is syntenic to a previously identified human amplicon at 11q13. Only one out of eight tumors without LOH at Chr 7 had twofold amplification; the other seven had no detectable amplification. Significant amplification is restricted to the chromosome with the Hras1 mutation. Gene amplification occurred without overexpression since only one of five tumors with amplification and one of six tumors without Ccnd1 amplification expressed increased protein. Although amplification of 11q13 occurs rather frequently in human tumors, 4NQO-induced oral cavity tumors in inbred mice are the first example of a murine tumor with consistent amplification. Our observations are strikingly similar to human head and neck SCC where overexpression of genes within the 11q13 amplicon is inconsistently detected. The amplification of genes localized to human 11q13 and the syntenic region on murine Chr 7 during tumorigenesis suggests that similar structural elements are present which predispose these regions to amplification during malignant transformation.