Molecular mechanisms of cytochrome c biogenesis: three distinct systems

The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c-type cytochromes. The hallmark of c-type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys-Xxx-Yyy-Cys-His signature motif). From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein. In this review, common principles of assembly for all systems and the molecular mechanisms predicted for each system are summarized. Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction. Accessory proteins of systems I and II co-ordinate the positioning of these two substrates at the membrane surface for covalent ligation. The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates. For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.     

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

Cowpea cooking characteristics as affected by micronisation temperature: a study of the physicochemical and functional properties of starch

Bechuana white cowpeas were micronised to three temperatures (130, 153 and 170 °C). Cooking properties of the cowpea seeds and the role of starch‐related properties were studied. In all cases, micronisation significantly reduced the cooking time and thus the time required for the cowpea seeds to attain a suitably soft texture. This was attributed in part to the significant improvement in rate of water absorption during cooking and starch pre‐gelatinisation, as evidenced by loss of birefringence and increased susceptibility of the cowpea starch to α‐amylase digestion. However, micronisation to 170 °C resulted in a severe deterioration in pasting properties of the cowpea flour, possibly due to starch depolymerisation and/or amylose‐associated crosslinking. Owing to these changes, cowpea seeds micronised to 170 °C required a longer cooking time than the other two micronised samples. Flour prepared from cowpea seeds micronised to 170 °C may have limited starch functionality. Copyright © 2006 Society of Chemical Industry     

18th World Congress, member assembly 1996 and professional board meeting of Rehabilitation International (RI) 9/13-9/20 in Auckland, New Zealand

Anterior knee pain in our military population consititutes a diagnostic and therapeutic problem that is not always easily solved. In recent years, a better understanding of the biomechanics of the patello-femoral joint has contributed to the successful classification and treatment of many patients. However, the cause of pain remains elusive in a significant subset of patients who show no clinical or radiographic evidence of patellar malalignment and continue to have persistent pain despite adequate physiotherapy. In this article, the classification and approach to anterior knee pain are reviewed. Emphasis is placed on dividing patients into four subsets namely; normal tracking, patellar tilt, patellar subluxation and extra-articular causes of anterior knee pain. A more rational approach to the problem can then be employed and successful rehabilitation and/or surgery can be expected.     

Cracking of concrete structures due to imposed strains with regard to design of reinforcement

The principles for control of cracking in concrete structures due to imposed strain are investigated in this paper. The effects of uniform and non-uniform cooling and shrinkage are studied in medium-thick walls with regard to the amount of reinforcement necessary to distribute cracks. The influence of reinforcement on the behavior after the first crack is investigated, in particular its ability to reduce crack widths. The restraint force is significantly reduced for non-uniformly distributed imposed strain due to softening and cracking in the concrete. The amount of minimum reinforcement needed to distribute cracks is therefore significantly smaller for imposed strain in the form of drying shrinkage than for thermal imposed strain. Crack reinforcement is most effective for structures with large dimensions in the direction of external restraint.     

Determination of the biological activity of heparin by use of a chromogenic substrate

We describe a new method for determining the biological activity of heparin in plasma with use of thrombin and the substrate Tos-Gly-Pro-Arg-pNA. The procedure is based on the photometric determination of the inactivation of thrombin after incubation with plasma in the presence of endogenous antithrombin III (At III). The method allows the specific determination of heparin concentrations from 0.02 USP to 0.8 USP/ml of plasma in the presence of normal At III levels. It has been carried out manually by use of an Eppendorf spectrum line photometer or automatically by use of a Vitatron Akes analyzer. For evaluation, the results were compared with two standard samples which contained heparin in the low and high therapeutic range, respectively.