Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

Bivariate optimization of pedalling rate and crank arm length in cycling

The contribution of this paper is a bivariate optimization of cycling performance. Relying on a biomechanical model of the lower limb, a cost function derived from the joint moments developed during cycling is computed. At constant average power, both pedalling rate (i.e. rpm) and crank arm length are systematically varied to explore the relation between these variables and the cost function. A crank arm length of 170 mm and pedalling rate of 100 rpm correspond closely to the cost function minimum. In cycling situations where the rpm deviates from 100 rpm, however, crank arms of length other than 170 mm yield minimum cost function values. In addition, the sensitivity of optimization results to both increased power and anthropometric parameter variations is examined. At increased power, the cost function minimum is more strongly related to the pedalling rate, with higher pedalling rates corresponding to the minimum. Anthropometric parameter variations influence the results significantly. In general it is found that the cost function minimum for tall people occurs at longer crank arm lengths and lower pedalling rates than the length and rate for short people.     

Comparative permeability of canine visceral and parietal pleura

To determine the permeability of canine pleural mesothelium, visceral and intercostal parietal pleura from mongrel dogs was carefully stripped from the underlying tissue and mounted as a planar sheet in a Ussing-type chamber. The hydraulic conductivity (Lp) was determined from the rate of volume flux in response to hydrostatic pressure gradients applied to either the mucosal or serosal surface of the pleural membrane. The diffusional permeability (Pd) of radiolabeled water, sucrose, inulin, and albumin was determined under equilibrium conditions from the unidirectional tracer flux. The Lp of the visceral pleura was 0.39 +/- 0.032 (SE) X 10(-4) ml.s-1.cmH2O-1.cm-2 and that Lp of parietal pleura was 1.93 +/- 0.93 X 10(-4) ml.s-1.cmH2O-1.cm-2 (P less than 0.001). The Pd of the visceral pleura ranged from 12.21 +/- 0.45 X 10(-4) cm/s for 3H2O to 0.34 +/- 0.03 X 10(-4) cm/s for [3H]albumin. The Pd of the parietal pleura for water and sucrose was similar to that of the visceral membrane, whereas its Pd for the larger inulin and albumin molecules was greater than that of visceral pleura (P less than 0.01). A spontaneous potential difference could not be detected across either membrane. The relatively higher parietal pleural Lp and Pd for larger solutes is probably due to the presence of stomata in this membrane. These results indicate that both the parietal and the visceral pleura are extremely permeable tissues which offer little resistance to water and solute flux.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

Cell type-specific gene expression in the Drosophila melanogaster male accessory gland.

The accessory gland of the male Drosophila melanogaster plays a vital role in reproduction. This secretory organ synthesizes products that are transferred to the female and are necessary to elicit the proper physiological and behavioral responses in the female. The accessory gland is composed of two morphologically distinct secretory cell types, the main cells and the secondary cells. Previous studies identified some genes expressed in main cells or in all accessory gland cells. In this paper we use P-element mediated enhancer traps to examine gene expression in the accessory gland. We show that, in addition to genes expressed in main cells only or in all accessory gland secretory cells, there are genes expressed specifically in secondary cells. Each cell type is uniform in the expression of its genes. Our results demonstrate that the two cell types are not only morphologically distinct but also biochemically distinct. We also show that the two cell types differ in their regulation of gene expression in response to mating activity.     

Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.     

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.     

Phenolsulphonphthalein conjugation and biliary excretion in the rat: influence of phenobarbital and 3-methylcholanthrene

The effect of phenobarbital and 3-methylcholanthrene pretreatment on the biliary excretion of phenolsulphonphthalein (PSP) was investigated in male Wistar rats. The dye was injected at a single dose of 200 mumol/kg body wt. About 20% of the compound was excreted as a glucuronide in the controls, the liver UDP-glucuronyltransferase activity toward PSP being 0.064 +/- 0.005 nmol.min-1.mg protein-1. Treatment for two weeks with phenobarbital (354 mumol.kg body wt-1.day-1) caused a transient increase in conjugated and unconjugated PSP excretion, but glucuronyltransferase activity was not modified. 3-Methylcholanthrene pretreatment for 4 days (75 mumol.kg body wt-1.day-1) also enhanced biliary excretion of the dye, but the increase corresponded only to the glucuronide and glucuronyltransferase activity was significantly enhanced by 20%. Our data indicate that not only the rate of biotransformation but also other factors could be responsible for increased PSP biliary excretion following administration of microsomal enzyme inducers.