Free fatty acids as inhibitors of the respiratory chain in testicular homogenates

The supernatants of the 440 000 . g . min centrifugation of homogenates of rat, bull and boar testicles and sperm inhibit the NADH-oxidase activity of non-phosphorylating submitochondrial particles (ETP). Whereas no inhibitory activity was observed with young rats (150 g), a marked inhibition was detected with heavier animals. The inhibitory activity of testicles was located in the microsomal fraction. The reaction of the testicular inhibitor with the ETP is initiated by an instant reversible binding followed by a slow irreversible inhibition of the electron transport. The reason of the time-dependence is neither an interaction between the enzymes of the ETP and those of the microsomal electron transport nor a slow degradation of the ETP by microsomal phospholipases. Some observations indicate an indirect involvement of phospholipase via the formation of free fatty acids (FFA). The inhibitory fraction could be solubilized from the microsomes both by sodium cholate treatment and by ethanol extraction. After separation of the lipid classes by chromatography on silica gel and gas-chromatographic analysis the inhibitory fraction was identified as a mixture of free fatty acids (FFA) of different chain lengths and degree of saturation. Thus a new effect of FFA on the mitochondrial electron transport has been detected which is different from other actions known up till now. The degradation of the phospholipids of the endoplasmic reticulum in the spermatozoa may be the source of the enhanced formation of FFA. An inhibition of the cell respiration presumably does not occur in vivo. The high FFA level in the testicular homogenates of sexually mature animals is a consequence of an intensive FFA metabolism, especially of high phospholipase activity.     

Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells

We have cultured myogenic cells derived from primary explants and a cell line (L6) in a lipid-depleted medium (LDM) and produced large alterations of the fatty acyl and polar headgroup composition and of the cellular sterol levels. These alterations were produced by altering the composition of the media as follows: removing biotin and providing exogenous fatty acid; removing choline and providing exogenous ethanolamine or choline analogues; and by adding 25-OH cholesterol, an inhibitor of 3-hydroxy-3-methylglutarate (HMG)-CoA reductase. Relatively small, secondary alterations of other lipid classes accompany the large primary alteration. In general, they are not obviously compensatory for the primary alteration by retaining some physical property. We have explored the influence of these lipid alterations on myoblast proliferation and fusion into myotubes. In general, considerable variability appears tolerated, but there also appear to be limits. Long-term cultures grown in media containing a single fatty acid do not proliferate indefinitely, and the fatty acid does not become the sole fatty acyl component of the phospholipids. This phenomenon is also observed for cultures enriched in phosphatidylethanolamine (PE) or phosphatidyldimethylethanolamine (PDME). The influence of the lipid alterations on fusion is particularly interesting. The inclusion of 25-OH cholesterol inhibits fusion. Enrichment of the fatty acyl chains with elaidate or the polar headgroups with PE also inhibits fusion, but in contrast to that by 25-OH cholesterol, a significant fraction of the myoblasts are aligned and interacting with each other. Oleate enrichment enhances the rate of fusion.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

Analysis of two different tandem repetitive elements within the human apolipoprotein B gene.

A large number of copies of the sequence (dTG-dAC)n, where n is between 10 and 60, exist in the human genome, and many are useful as polymorphic markers. One of these sequences occurs about 3 kilobases 5' of the human apolipoprotein (apo) B gene as seven distinguishable alleles containing from (TG)12 to (TG)18. This repeat is also present in the DNA of other primates. A second alternating purine-pyrimidine sequence with nine dinucleotide repeats and located in intron 4 is not polymorphic. Together with the apoB hypervariable repeat immediately 3' of the gene, the (TG)n sequence will provide a useful haplotype marker capable of distinguishing a large number of human apoB alleles, some of which may be associated with disease states.     

Elucidation of the complete Azorhizobium nicotinate catabolism pathway.

A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.     

Polymorphism of human glycoprotein Ib alpha results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. Structure/function implications.

Four polymorphic variants of the platelet receptor for von Willebrand factor, glycoprotein Ib, have been described that differ in molecular weight on sodium dodecyl sulfate-polyacrylamide gels (Moroi, M., Jung, S. M., and Yoshida, N. (1984) Blood 64, 622-629). A recent report localized the polymorphic site to the heavily O-glycosylated region of the glycoprotein Ib alpha-chain known as the macroglycopeptide (Meyer, M., and Schellenberg, I. (1990) Thromb. Res. 58, 233-242). This region contains several tandem repeats of a mucin-like sequence, which appeared to be a likely site for polymorphic variation. We amplified genomic DNA corresponding to the macroglycopeptide from 206 individuals from four ethnic groups and identified three length variants based on the migration of the amplified DNA on denaturing polyacrylamide gels. DNA sequencing revealed that the three variants represented four alleles, two of which varied by only one base pair, a difference that did not result in an amino acid change. The three length variants differed in the number of tandem repeats of a 39-base pair sequence that results in perfect duplication of a 13-amino acid sequence that originated within a region flanked by Glu-396 and Thr-411. The smallest isoform contained one such sequence; the next largest, two repeats; and the largest, three repeats. The DNA sequence containing the tandem repeats was flanked by direct repeats typical of the target site duplications found flanking transposed DNA, suggesting a mechanism for acquisition of this region by the primordial glycoprotein Ib alpha precursor. The amino acid sequence of the repeated element that accounts for the polymorphism contained five sites for potential O-glycosylation, which together with the repeated amino acids would result in incremental differences in molecular weight of approximately 6,000 between the different isoforms. The addition of repeats to the macroglycopeptide is predicted to increase the length of this elongated glycosylated region and extend the distance between the ligand-binding domain of glycoprotein Ib and the platelet plasma membrane, an effect that would project the ligand-binding domain farther into the bloodstream. Such a change may alter the susceptibility of platelets to shear-induced activation, a process that requires an interaction between glycoprotein Ib and von Willebrand factor.     

Genetic control and expression of the major ejaculatory bulb protein (PEB-me) inDrosophila melanogaster

PEB-me is a predominant protein of matureDrosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35–39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200–800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded bypeb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant fortra, tra-2, dsx, orix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB inDrosophila reproduction are discussed.     

Effect of lung volume on plateau response of airways and tissue to methacholine in dogs.

We have recently shown in dogs that much of the increase in lung resistance (RL) after induced constriction can be attributed to increases in tissue resistance, the pressure drop in phase with flow across the lung tissues (Rti). Rti is dependent on lung volume (VL) even after induced constriction. As maximal responses in RL to constrictor agonists can also be affected by changes in VL, we questioned whether changes in the plateau response with VL could be attributed in part to changes in the resistive properties of lung tissues. We studied the effect of changes in VL on RL, Rti, airway resistance (Raw), and lung elastance (EL) during maximal methacholine (MCh)-induced constriction in 8 anesthetized, paralyzed, open-chest mongrel dogs. We measured tracheal flow and pressure (Ptr) and alveolar pressure (PA), the latter using alveolar capsules, during tidal ventilation [positive end-expiratory pressure (PEEP) = 5.0 cmH2O, tidal volume = 15 ml/kg, frequency = 0.3 Hz]. Measurements were recorded at baseline and after the aerosolization of increasing concentrations of MCh until a clear plateau response had been achieved. VL was then altered by changing PEEP to 2.5, 7.5, and 10 cmH2O. RL changed only when PEEP was altered from 5 to 10 cmH2O (P < 0.01). EL changed when PEEP was changed from 5 to 7.5 and 5 to 10 cmH2O (P < 0.05). Rti and Raw varied significantly with all three maneuvers (P < 0.05). Our data demonstrate that the effects of VL on the plateau response reflect a complex combination of changes in tissue resistance, airway caliber, and lung recoil.     

Lung tissue behavior during methacholine challenge in rabbits in vivo.

Previous studies have shown that lung challenge with smooth muscle agonists increases tissue viscance (Vti), which is the pressure drop between the alveolus and the pleura divided by the flow. Passive inflation also increases Vti. The purpose of the present study was to measure the changes in Vti during positive end-expiratory pressure- (PEEP) induced changes in lung volume and with a concentration-response curve to methacholine (MCh) in rabbits and to compare the effects of induced constriction vs. passive lung inflation on tissue mechanics. Measurements were made in 10 anesthetized open-chest mechanically ventilated New Zealand male rabbits exposed first to increasing levels of PEEP (3-12 cmH2O) and then to increasing concentrations of MCh aerosol (0.5-128 mg/ml). Lung elastance (EL), lung resistance (RL), and Vti were determined by adjusting the equation of motion to tracheal and alveolar pressures during tidal ventilation. Our results show that under baseline conditions, Vti accounted for a major proportion of RL; during both passive lung inflation and MCh challenge this proportion increased progressively. For the same level of change in EL, however, the increase in Vti was larger during MCh challenge than during passive inflation; i.e., the relationship between energy storage and energy dissipation or hysteresivity was dramatically altered. These results are consistent with a MCh-induced change in the intrinsic rheological properties of lung tissues unrelated to lung volume change per se. Lung tissue constriction is one possible explanation.     

Immunotherapy for remission maintenance in acute myeloblastic leukemia

Summary Forty-eight patients with acute myeloblastic leukemia in remission were treated with immunotherapy in addition to remission-maintenance chemotherapy. The first 16 patients were treated with weekly BCG and a leukemia cell vaccine (group 1). The next 32 patients were randomly allocated to receive BCG and a leukemia cell vaccine given once monthly (group 2) or BCG given monthly with no leukemia cell vaccine (group 3). There was no significant difference in remission duration or survival between the randomly allocated groups (2 and 3).Comparisons with group 1 are limited by the non-random allocation to this group, but selection bias was unlikely and clinical features were similar in the three patient groups. No significant difference in remission duration or survival was seen amongst the three groups studied. There was no advantage in the addition of leukemia cell vaccine (groups 1 and 2) to BCG alone (group 3) and no advantage to weekly (group 1) versus monthly immunotherapy (groups 2 and 3). Only 7 of the 48 patients achieved a second remission, and 4 of these were short-term partial remissions.The following are contributing members of the Toronto Leukemia Study Group: Doctor's Hospital, Harvey Silver MD; Humber Memorial Hospital, Alan Seidenfeld MD; Mississauga Hospital, Michael King MD; Mount Sinai Hospital, Dominic Amato MD; Northwestern Hospital, Wilhelm Kwant MD; Oshawa General Hospital, Hak Chiu MD; St Michael's Hospital, Bernadette Garvey MD, Kenneth Butler MD; St Joseph's Hospital, H. James Watt MD, Murray Davidson MD; Toronto General Hospital, Gerald Scott MD, William Francombe MD, Kenneth Shumak MD; John Crookston MD, PhD; Toronto Western Hospital, James G. Watt MD, David Sutton MD; Michael Baker MD; Domenic Pantalony MD; Wellesley Hospital, Dale Dotten MD; Women's College Hospital, George Kutas MD; York Finch Hospital, Sam Berger MD