Human dynein and sperm pathology

Human spermatozoa with normal structure and with different axonemal deficiencies (absence of axoneme, of arms, or of central structures) were studied by electron microscopy, SDS-polyacrylamide gel electrophoresis, and ATPase activity measurements. Normal human sperm possess a complement of high molecular weight polypeptides with an electrophoretic migration similar to that of sea urchin and other mammalian sperm dyneins. Human high molecular weight bands are numbered one to four in order of increasing of electrophoretic mobility; all of them are absent in spermatozoa that lack axoneme. The absence of doublet arms, coincides with the absence of bands 2, 3, and 4; the absence of central structures coincides with a reduction in intensity of band 2. In the latter two abnormal conditions, band 1 has an increased intensity. The data are tentatively interpreted by attributing the polypeptides forming bands 3 and 4 to the arm structure, whereas band 2 is supposed to contain a mixture of polypeptides localized in the arms and in the central structures; these abnormal sperm contain modified polypeptides which gather in band 1. Histochemical ATPase stainings indicate that this enzyme is localized mainly in the doublet arms and, to a minor extent, in the central structures.     

Nutrient and chlorophyll a temporal patterns in eutrophic mountain ponds with contrasting macrophyte coverage

Hydrobiologia - In shallow lakes, macrophytes have important effects on food webs, community structure and nutrient dynamics. For this reason they play a significant role in the restoration of...     

Lymphocyte transformation and macrophage migration inhibition by electrofocused and gel-filtered fractions of group A streptococcal filtrate

Culture filtrates of group A streptococci were fractionated either by isoelectric focusing on a sucrose gradient at pH 3–10, or by gel filtration on a G-75 Superfine Sephadex column. Some fractions induced lymphocyte transformation, others inhibition of macrophage migration, and others both. With the two types of fractionation here used the lymphocyte transformation activity was concentrated in a single peak, while the activity responsible for macrophage migration inhibition was scattered over multiple fractions. The significance of these findings is discussed.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Molecular evidence of triplication in the haptoglobin Johnson variant gene

Summary The protein and gene structure of the Hp Johnson variant (Hp3) were analyzed in two related heterozygous individuals. The molecular weight (23kd) and amino acid composition of Hp3 alpha chain were in agreement with the triplicated structure first suggested by Smithies in 1964. Direct gene analysis by Southern blotting showed a three-fold tandem repeat of the same 1.7 kb DNA segment implicated in the Hp2 gene duplication. On the basis of these data a nine exon model for the Hp3 gene is proposed.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.