alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling

Soluble transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP)-binding proteins are widely distributed in mammalian tissues and control cytokine access to membrane signaling receptors. The serum and bone-resident glycoprotein alpha2-HS-glycoprotein/fetuin (ASHG) binds to TGF-beta/BMP cytokines and blocks TGF-beta1 binding to cell surface receptors. Therefore, we examined bone growth and remodeling phenotypes in ASHG-deficient mice. The skeletal structure of Ahsg(-/-) mice appeared normal at birth, but abnormalities were observed in adult Ahsg(-/-) mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age in Ahsg(-/-) mice. However, bone formation was increased in Ahsg(-/-) mice as indicated by greater cortical thickness, accelerated trabecular bone remodeling, and increased osteoblast numbers on bone surfaces. The normal age-related increase in cortical thickness and bone mineral density was accelerated in Ahsg(-/-) mice and was associated with increased energy required to fracture. Bone formation in response to implanted BMP cytokine extended further from the implant in Ahsg(-/-) compared with Ahsg(+/+) mice, confirming the interaction between ASHG and TGF-beta/BMP cytokines in vivo. Our results demonstrate that ASHG blocks TGF-beta-dependent signaling in osteoblastic cells, and mice lacking ASHG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis.     

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section

Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.     

Human Mucin Genes Assigned to 11p15.5: Identification and Organization of a Cluster of Genes

Four distinct genes that encode mucins have previously been mapped to chromosome 11p15.5. Three of these genes (MUC2, MUC5AC,andMUC6) show a high level of genetically determined polymorphism and were analyzed in the CEPH families. Linkage analysis placed all three genes on the genetic map in a cluster betweenHRASandINS,and more detailed analysis of recombinant breakpoints revealed thatMUC6is telomeric toMUC2.Using these recombinantsD11S150was mapped close toMUC2.Ten of the 11 recombinant chromosomes studied in detail were paternal, and the recombinant events were distributed throughout the 11p15 region, suggesting that the high level of recombination observed in 11p15.5 is not due to a particular recombinational hot spot. Pulsed-field gel electrophoresis was used to make a detailed physical map of theMUCcluster and to integrate the physical and genetical maps. The gene order was determined to beHRAS–MUC6–MUC2–MUC5AC–MUC5B–IGF2.TheMUCgenes span a region of some 400 kb and the map extends 770 kb and contains 15 putative CpG islands. The order of theMUCgenes on the map corresponds to the relative order of their expression along the anterior–posterior axis of the body, suggesting a possible functional significance to the gene order.     

Lactase polymorphism in adult British natives: estimating allele frequencies by enzyme assays in autopsy samples.

In an attempt to estimate allele frequencies of lactase persistence in adult British natives, sucrase was assayed simultaneously with lactase under conditions that gave optimal activities for both enzymes. A trimodal distribution in the ratios of enzyme activities was demonstrated. Circumstantial evidence and statistical analyses suggest that the trimodal distribution is due to the different levels of lactase activity in the three genotypes--homozygous persistent, heterozygous, and homozygous nonpersistent, and that it is possible to correct for "nongenetic" variation by using sucrase as an internal standard. The allele frequency for lactase persistence was estimated to be .747. The implications of our findings on the genetic mechanisms involved in lactase persistence are discussed.     

The one-electron reduction potentials of NAD

The one-electron reduction potential (E¹₇) of NAD⁺ has been determined by pulse radiolysis to be ?0.94 V. E²₇ (E¹₇ for the free radical, NAD^.) is +0.30 V. E¹₇ for 1-methylisonicotinamide is ?0.77 V.     

Aerial performance of stalk-eyed flies that differ in eye span

Stalk-eyed flies have eyes placed laterally away from the head on elongated peduncles. The elongation of eye span may increase the energetic cost of flight, reduce flight performance via aerodynamic effects or via increased load, or necessitate compensatory changes in other body dimensions. Body mass and body dimensions were measured to test the hypothesis that elongation of eye span is correlated with increased head mass in two closely related species of stalk-eyed flies. Cyrtodiopsis whitei is sexually dimorphic, with the eye span of larger males exceeding body length. Cyrtodiopsis quinqueguttata is sexually monomorphic with eye span substantially less than body length. Although eye span was significantly longer in C. whitei, head mass did not differ between species after accounting for differences in body mass. C. whitei males had longer wings, heavier thoraxes, and lighter abdomens in relation to body mass than did female C. whitei or C. quinqueguttata of either sex. Three-dimensional tracking of flight paths showed that path velocity and the horizontal component of velocity did not differ according to species or sex, but the long-eyed C. whitei males showed reduced overall aerial performance by flying at shallower ascent angles and reduced vertical velocity. Although increased mass loading does not occur in C. whitei males, increased drag, aerodynamic effects from the wake of the eye stalks, and constrained visual processing are possible mechanisms which could cause their reduced performance. Accepted: 7 June 2000     

A novel technique for nonvolitional assessment of quadriceps muscle endurance in humans.

Assessment of quadriceps endurance is of interest to investigators studying human disease. We hypothesized that repetitive magnetic stimulation (rMS) of the intramuscular branches of the femoral nerve could be used to induce and quantify quadriceps endurance. To test this hypothesis, we used a novel stimulating coil to compare the quadriceps endurance properties in eight normal humans and, to confirm that the technique could be used in clinical practice, in eight patients with advanced chronic obstructive pulmonary disease (COPD). To validate the method, we compared in vivo contractile properties of the quadriceps muscle with the fiber-type composition and oxidative enzyme capacity. We used a Magstim Rapid(2) magnetic nerve stimulator with the coil wrapped around the quadriceps. Stimuli were given at 30 Hz, a duty cycle of 0.4 (2 s on, 3 s off), and for 50 trains. Force generation and the surface electromyogram were measured throughout. Quadriceps twitch force, elicited by supramaximal magnetic stimulation of the femoral nerve, was measured before and after the protocol. Quadriceps muscle biopsies were analyzed for oxidative (citrate synthase, CS) and glycolytic (phosphofructokinase, PFK) enzyme activity and myosin heavy chain isoform protein expression. The time for force to fall to 70% of baseline (T(70)) was shorter in the COPD group than the control group: 55.6 +/- 26.0 vs. 121 +/- 38.7 s (P = 0.0014). Considering patients and controls together, positive correlations were observed between T(70) and the proportion of type I fibers (r = 0.68, P = 0.004) and CS-to-PFK ratio (CS/PFK) (r = 0.67, P = 0.005). We conclude that quadriceps endurance assessed using rMS is feasible in clinical studies.     

Effects of voluntary activity and genetic selection on muscle metabolic capacities in house mice Mus domesticus.

Selective breeding is an important tool in behavioral genetics and evolutionary physiology, but it has rarely been applied to the study of exercise physiology. We are using artificial selection for increased wheel-running behavior to study the correlated evolution of locomotor activity and physiological determinants of exercise capacity in house mice. We studied enzyme activities and their response to voluntary wheel running in mixed hindlimb muscles of mice from generation 14, at which time individuals from selected lines ran more than twice as many revolutions per day as those from control (unselected) lines. Beginning at weaning and for 8 wk, we housed mice from each of four replicate selected lines and four replicate control lines with access to wheels that were free to rotate (wheel-access group) or locked (sedentary group). Among sedentary animals, mice from selected lines did not exhibit a general increase in aerobic capacities: no mitochondrial [except pyruvate dehydrogenase (PDH)] or glycolytic enzyme activity was significantly (P < 0.05) higher than in control mice. Sedentary mice from the selected lines exhibited a trend for higher muscle aerobic capacities, as indicated by higher levels of mitochondrial (cytochrome-c oxidase, carnitine palmitoyltransferase, citrate synthase, and PDH) and glycolytic (hexokinase and phosphofructokinase) enzymes, with concomitant lower anaerobic capacities, as indicated by lactate dehydrogenase (especially in male mice). Consistent with previous studies of endurance training in rats via voluntary wheel running or forced treadmill exercise, cytochrome-c oxidase, citrate synthase, and carnitine palmitoyltransferase activity increased in the wheel-access groups for both genders; hexokinase also increased in both genders. Some enzymes showed gender-specific responses: PDH and lactate dehydrogenase increased in wheel-access male but not female mice, and glycogen phosphorylase decreased in female but not in male mice. Two-way analysis of covariance revealed significant interactions between line type and activity group; for several enzymes, activities showed greater changes in mice from selected lines, presumably because such mice ran more revolutions per day and at greater velocities. Thus genetic selection for increased voluntary wheel running did not reduce the capability of muscle aerobic capacity to respond to training.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.