Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L: Involvement of Lipid-linked Intermediates

Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[¹⁴C]N-acetylglucosamine and GDP-[¹⁴C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue.     

Membrane-associated Glycosyl Transferases in Cotyledons of Pisum sativum: Differential Effects of Magnesium and Manganese Ions

In crude particulate fractions isolated from pea (Pisum sativum) cotyledons, the transfer of radioactivity from GDP-[¹⁴C]mannose to glycolipid appears to be preferentially stimulated by Mn²⁺ while the transfer to lipid-free residue is enhanced by Mg²⁺. In contrast, the transfer of radioactivity from UDP-N-acetyl-[¹⁴C]glucosamine to glycolipid shows preferential stimulation by Mg²⁺ while the transfer to lipid-free residue prefers Mn²⁺. These results are accounted for by the differential stimulation by Mg²⁺ and Mn²⁺ of glycosyl transferases associated with subcellular membranes which were separated by isopycnic sucrose density centrifugation.     

Digital image analysis of AgNORs in cervical smears of women with premalignant and malignant lesions of the uterine cervix

We performed a hospital-based, unmatched case-control study to investigate the association between progressive stages of cervical neoplasia and digital analysis of cell proliferation by silver stained nucleolus organizer region associated proteins (AgNORs). We measured cell proliferation levels in the cervical epithelial cells of 10 women with low grade squamous intraepithelial lesions (LG-SIL), eight with high grade squamous intraepithelial lesions (HG-SIL), 11 with cervical cancer (CC) and eight with no cervical lesions (controls) using the AgNORs technique. Cell proliferation was measured by digital image analysis (DIA). DIA revealed increased total areas of AgNORs in HG-SIL and CC compared to LG-SIL and control patients. AgNORs with a kidney or cluster shape exhibited greater areas than those with a spherical or long shape. We propose a cut-off of 118 pixels to differentiate benign (control and LG-SIL) from malignant (HG-SIL and CC) lesions. DIA of AgNORs is a simple and inexpensive method for studying proliferation. The increased total area of AgNORs in malignant lesions provides information regarding cell behavior and may be related to cervical carcinogenesis; however, further validation studies are required to establish its usefulness in cytological analysis.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.