Understanding the musculoskeletal injury risk of women in combat: the effect of infantry training and sex on musculoskeletal injury incidence during British Army basic training.

INTRODUCTION: Until recently, women were excluded from British combat roles. Their risk for musculoskeletal injury during basic training is two to three times higher than men. To better understand the musculoskeletal injury risk of women in British Army infantry basic training, we compared injury incidence between (1) men in standard entry training and men in infantry training, to assess the risk of infantry training; and (2) men and women in both standard entry and officer basic training, to assess the risk in women compared with men. METHODS: The incidence of musculoskeletal injury was determined from defence medical records for all men entering infantry training, and for all men and women entering standard entry and officer training, between April 2015 and March 2016. RESULTS: 7390 men (standard entry, n=4229; infantry, n=2683; officer, n=478) and 696 women (standard entry, n=626; officer, n=70) entered basic training. Men in infantry training had a lower incidence of musculoskeletal injury (391 vs 417 per 1000 personnel, OR 0.90 (95% CI 0.81 to 0.99), p=0.028) and a higher incidence of stress fracture (14 vs 5 per 1000 personnel, OR 2.80 (95% CI 1.64 to 4.80), p<0.001) than men in standard entry training. Women had a higher incidence of musculoskeletal injury than men in standard entry training (522 vs 417 per 1000 personnel, OR 1.53 (95% CI 1.29 to 1.81), p<0.001) and a higher incidence of stress fracture than men in officer training (114 vs 19 per 1000 personnel, OR 6.72 (95% CI 2.50 to 18.07), p<0.001). CONCLUSION: Women in infantry training may be at similar risk for musculoskeletal injury, but at higher risk for stress fracture, compared with their non-infantry counterparts. Women in infantry training may be at higher risk for musculoskeletal injury and stress fracture compared with men in infantry training.

Avian agnosia: A window into auditory semantics.

The functional and neural organisation of auditory knowledge is relatively poorly understood. The breakdown of conceptual knowledge in semantic dementia has revealed that pre-morbid expertise influences the extent to which knowledge is differentiated. Whether this principle applies to a similar extent in the auditory domain is not yet known. Previous reports of patients with impaired auditory vs. intact visual expert knowledge suggest that expertise may have differential effects upon the organisation of auditory and visual knowledge. An equally plausible alternative, however, is that auditory knowledge is simply more vulnerable to deterioration. Thus, expertise effects in the auditory domain may not yet have been observed because knowledge of auditory expert vs. non-expert knowledge has yet to be compared. We had the opportunity to address this issue by studying SA, a patient with semantic dementia and extensive pre-morbid knowledge of birds. We undertook a systematic investigation of SA's auditory vs. visual knowledge from matched expert vs. non-expert categories. Relative to a group of 10 age, education and IQ matched bird experts, SA showed impaired auditory vs. intact visual avian knowledge, despite intact basic auditory perceptual abilities. This was explained by independent effects of modality and expertise. Thus, he was also disproportionately impaired for auditory vs. visual knowledge of items from non-expert categories. In both auditory and visual modalities, his performance was relatively more impaired on tests of non-expert vs. expert knowledge. These findings suggest that, while auditory knowledge may be more vulnerable to deterioration, expertise modulates visual and auditory knowledge to a similar extent.

The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes.

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 1/2 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1 was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physiochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.

Role of N-terminal domain of histidine-rich glycoprotein in modulation of macrophage Fc gamma receptor-mediated phagocytosis.

A brief exposure of murine peritoneal inflammatory macrophages to plasma histidine-rich glycoprotein (HRG; 77,000-81,000 MW) for 1-2 hr increased Fc gamma receptor (Fc gamma R) expression and phagocytic function in these cells. However, a continual culture of the cells without the presence of HRG for the next 18-48 hr resulted in down-regulation of Fc gamma R expression and phagocytic function. Similarly, HRG decreased Fc gamma RII expression in less differentiated human THP-1 monocytic cells during treatment for 18 hr, as determined by cellular ELISA and metabolic labelling. The molecular mechanism by which HRG regulates Fc gamma R expression is unknown. However, at a relatively high concentration (> 1 microgram/ml), HRG altered the cellular metabolism by increasing cellular protein synthesis but reducing protein secretion. These observations suggest a likely mechanism for the HRG-mediated reduction of Fc gamma R expression. A degraded HRG (40,000 MW) which possessed an identical N-terminal sequence as that of the native HRG was capable of decreasing macrophage Fc gamma R expression and phagocytosis. The results indicate that the functional domain of HRG responsible for binding to macrophages is localized to the N-terminal half.

Adsorption and helical coiling of amphipathic peptides on lipid vesicles leads to negligible protection from cathepsin B or cathepsin D.

The processing of antigenic peptides for presentation by MHC molecules to T cells, may depend upon the function of a second, consensus sequence in or near the T cell-presented epitope. One such processing-regulating sequence appears to be composed of amino acids Leu, Ile, Val, Phe, and Met recurring in a fashion to form a longitudinal, hydrophobic strip when the excised peptide is coiled as an alpha-helix. Such a hydrophobic strip-of-helix may: (a) scavenge peptides from lumens onto lipid membranes of digestion vesicles, (b) stabilize peptides there as protease-resistant helices, (c) specify recognition by the antigenic peptide-binding sites of chaperonin proteins, transmembranal transporters, or MHC molecules. By circular dichroism and electron paramagnetic resonance, we demonstrated that peptides with recurrent hydrophobic residues potentially forming longitudinal strips adsorbed to, and partially coiled as helices on, di-O-hexadecyl, D-L-alpha-phosphatidylcholine (DHPC) vesicles. Cathepsin B or cathepsin D cleavages of three such peptides were identified. With either enzyme, it made no significant difference whether a peptide substrate was in solution or bound to vesicles in terms of efficiency and specificity of peptide bond cleavages. We conclude that protease resistance, per se, of membrane-adsorbed, helically coiled peptides is not a major factor in the selection for T cell presentation of epitopes in peptides which have a motif with a longitudinal hydrophobic strip.

Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein.

Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.

Hypothesis for the control of clotting factor VIII inhibitory antibodies by decreasing potency of helper T-cell-recognized epitopes in factor VIII.

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes.

Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles.

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.

Comparison of ram sperm interaction with bovine cervical mucus.

Ram sperm penetration in estrous bovine cervical mucus was evaluated from ejaculates collected during long (16L:8D) and short (8L:16D) photoperiods with varying ambient temperatures. The distance traveled by vanguard sperm was affected by an interaction of the photoperiod and temperature (P<0.001). Sperm migration distance in a capillary tube filled with mucus (22.5 to 23.2 mm) was greater when sperm were collected from rams on short days and when the ambient temperature was between 10 and 31 degrees C than when sperm were collected under either long or short days (15.5 to 17.8 mm), when ambient temperatures were between 1 to 9 degrees C. Incidence of head-to-head agglutination of sperm differed by temperature (P<0.05) and photoperiodic (P<0.09) conditions. The percentage of ejaculates with evidence of sperm agglutination in the mucus was higher in long (62.5%) vs short (45.2) days, and it was greater in sperm collected in warm (61%) vs cold (44%) days. Physical interaction of cervical mucus with spermatozoa was examined. The binding of an iodinated protein from lyophilized mucus to a detergent soluble extract of washed or unwashed sperm was observed. These data show that both photoperiod and temperature affect the interaction of ram sperm with bovine cervical mucus.

Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control.

Glycogen synthase was purified from rat heart and muscle and electroblotted from sodium dodecyl sulfate polyacrylamide gels to polyvinylidene difluoride, and the NH2-terminal amino acid sequence was determined. The NH2-terminal amino acid sequence of the enzymes was identical. Further, phosphorylation site 2, a major cyclic AMP-dependent protein kinase recognition site in the rabbit muscle isozyme, is conserved in the rat isozymes suggesting that it serves an important function in hormonal regulation. However, two potentially important differences were observed. Threonine-5 and valine-8 of the rabbit muscle enzyme are serine and methionine residues, respectively, in the rat isozyme, the latter being critical in the analysis of phosphopeptides produced by cyanogen bromide cleavage. These variations may provide a partial explanation for previously observed differences in rat and rabbit phosphopeptide maps.

Further characterization of RLM2 and comparison with a related form of cytochrome P-450, RLM2b.

We have extended the characterization of RLM2, a constitutive form of rat liver cytochrome P-450, using immunochemical means to quantitate its presence in microsomes, to follow its development in maturing male and female rats, and to determine its response to prototypical P-450 inducers. In addition, RLM2 is compared to RLM2b, a form of P-450 with similar migration on SDS-PAGE and NH2-terminal amino acid sequence. RLM2b is expressed in both sexes at a level of 0.08 nmol/mg microsomal protein at 2 weeks of age. In female rats, this level is unchanged with maturation. However, in the male, the level declined with maturation to reach 0.02 nmol/mg protein by 12 weeks of age. RLM2 is a male-specific form of cytochrome P-450. Originally absent in the 2-week-old rat, it reached a level of 0.03 nmol/mg protein in the adult male, its appearance and increase coinciding with the onset of puberty. Both phenobarbital and 3-methylcholanthrene induced microsomal levels of RLM2b in the adult male and female rat. RLM2, however, was suppressed in the male rat, 58 and 42%, respectively, by the same treatments. RLM2b and RLM2 each catalyze a unique spectrum of hydroxytestosterone metabolites. RLM2b is highly site specific. In contrast, RLM2 produces several isomeric products in the same region of the testosterone molecule. Substitution of the acetyl group of progesterone for the 17-hydroxy group of testosterone did not alter the site specificity of RLM2b, but did alter it for RLM2, indicating, further, a difference in the active site conformation of the two enzymes. Although RLM2b and RLM2 responded differently to inducers and to a changing physiology during maturation, and were functionally quite distinct, the proteins showed a high degree of immunologic relatedness which is suggestive of significant structural similarities. Structural differences do exist, however, as alpha-chymotryptic digestion formed a number of peptide fragments that differed between the two proteins.

Isolation and analysis of murine serum amyloid P component cDNA clones.

In contrast to other animals, the biosynthesis of serum amyloid P component in mice is regulated as an acute-phase protein. As a first step in studying the regulation and biosynthesis of serum amyloid P component in the mouse, cDNA clones have been isolated from a liver cDNA library and sequenced. The largest of these clones was 960 bp in length, and contained an open reading frame encoding a protein of 224 amino acids. Comparison of the mouse cDNA sequence to that published for humans (Mantzouranis, E. C., S. B. Dowton, A. S. Whitehead, M. D. Edge, G. A. P. Bruns, and H. R. Colten, 1985. J. Biol. Chem. 260:7752.) revealed 74% identity for nucleotides in the translated region. Northern-blot analysis demonstrated that murine serum amyloid P component synthesis in the liver is directed by a 1.2-kb mRNA that is elevated in high responder (C57BL/6J) mice after thioglycollate-induced inflammation.

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae.

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).

A single receptor binds both insulin-like growth factor II and mannose-6-phosphate.

Amino acid sequences deduced from rat complementary DNA clones encoding the insulin-like growth factor II (IGF-II) receptor closely resemble those of the bovine cation-independent mannose-6-phosphate receptor (Man-6-P receptorCI), suggesting they are identical structures. It is also shown that IGF-II receptors are adsorbed by immobilized pentamannosyl-6-phosphate and are specifically eluted with Man-6-P. Furthermore, Man-6-P specifically increases by about two times the apparent affinity of the purified rat placental receptor for 125I-labeled IGF-II. These results indicate that the type II IGF receptor contains cooperative, high-affinity binding sites for both IGF-II and Man-6-P-containing proteins.

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Isolation of cDNA clones coding for rat isovaleryl-CoA dehydrogenase and assignment of the gene to human chromosome 15.

Rat liver mRNA encoding the cytoplasmic precursor of mitochondrial isovaleryl-CoA dehydrogenase was highly enriched by polysome immunopurification using a polyclonal monospecific antibody. The purified mRNA was used to prepare a plasmid cDNA library which was screened with two oligonucleotide mixtures encoding two peptides in the amino-terminal portion of mature rat isovaleryl-CoA dehydrogenase. Thirty-one overlapping cDNA clones, spanning a region of 2.1 kbp, were isolated and characterized. The cDNA sequence of a 5'-end clone, rIVD-13 (155 bp), predicts a mitochondrial leader peptide of 30 amino acid residues and the first 18 amino acids of the mature protein. These consecutive 18 residues completely matched the amino-terminal peptide determined by automated Edman degradation of the rat enzyme. The leader peptide contains six arginines, has no acidic residues, and is particularly rich in leucine, alanine, and proline residues. Southern blot analysis of DNAs from human-rodent somatic cell hybrids with an isolated rat cDNA (2 kbp) assigned the isovaleryl-CoA dehydrogenase gene to the long arm of chromosome 15, region q14----qter. The chromosomal assignment was confirmed and further refined to bands q14----q15 by in situ hybridization of the probe to human metaphase cells. This location differs from that of the gene for medium-chain acyl-CoA dehydrogenase, a closely related enzyme, which has been previously assigned to chromosome 1.