Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera,Culicidae)

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of spermoocyte interaction.     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.     

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.     

Repair of chemical carcinogen-induced damage in DNA of human lymphocytes and lymphoid cell lines--studies of the kinetics of removal of O6-methylguanine and 3-methyladenine

The removal of O6-methylguanine by human lymphoid cells corresponded, with certain assumptions, to a second-order chemical reaction in any given cell. There was a spectrum of proficiency in this respect for a considerable number of cells originating from different individuals and it was found that patients with diseases associated with autoimmunity tended to fall into the less proficient groups. E-B virus-induced lymphoid cell lines, derived from proficient, but not relatively deficient, peripheral blood lymphocytes, always (in 9/9 cases) reflected the level of proficiency of the donor lymphocytes with respect to removal of O6-methylguanine. Thus while proficient lymphocytes always produced proficient cell lines, deficient lymphocytes, in 3/8 cases, gave rise to more proficient cell lines. No evidence was found that groups of individuals exist who lack ability to remove 3-methyladenine from DNA, either from their blood lymphocytes or derived lymphoid cell lines.     

Long-term hyperalgesia in rats induced by neonatal administration of vasopressin antiserum

Vasopressin antiserum was given to two day old rats and the nociceptive thresholds were evaluated three months later. The rats were hypersensitive to pain when electrical current, but not heat, was used as the noxious stimulus. These animals were also insensitive to cold-water swim, a non-opioid form of stress analgesia. The vasopressin content in the pituitary or in the hypothalamus was not however modified by the neonatal treatment. The present results suggest a physiological role for vasopressin in non-opioid pain inhibitory systems.     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.