Detecting and minimizing zinc contamination in physiological solutions

Background  

Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors.     

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings

One bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar and residual dipolar couplings have been measured at sites in an ¹⁵N, ¹³C, 50% ²H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that ¹H-¹³C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the H_methylC_methylC angle are found to be small, with average differences on the order of 0.3 ± 0.1° and 0.4 ± 0.2° between CH₃, CH₂D and CH₃, CHD₂ isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.     

A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction

Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 x 10(8) and 1.1 x 10(13) vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 x 10(9) to 3.0 x 10(11) vg/mouse. Vector doses above 3.0 x 10(11) vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 x 10(12) vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 x 10(12) vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.     

Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.     

Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D

The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated. Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited. The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D. Re-evaluation of the potato inhibitor revealed that it too was more potent (>20-fold) towards yeast proteinase A than cathepsin D and so might be renamed the potato inhibitor of proteinase A. The potency towards yeast proteinase A may reflect a similarity between this fungal enzyme and aspartic proteinases produced by fungal pathogens which attack tomato and/or potatoes.     

Cytotoxic bromoindole derivatives and terpenes from the Philippine marine sponge Smenospongia sp

A detailed chemical analysis of a Philippine marine sponge Smenospongia sp. has been performed. This study yielded four new metabolites, 5-bromo-L-tryptophan (1), 5-bromoabrine (2), 5,6-dibromoabrine (3) and 5-bromoindole-3-acetic acid (4). The pyrroloiminoquinone alkaloid, makaluvamine O (5) as well as 5,6-dibromotryptamine (6), aureol (7) and furospinulosin 1 (8) were also isolated. Although 1 and 4 have been synthesized previously, this is the first report on the isolation of these compounds from a natural source. The furanosesterterpene furospinulosin 1 (8) was obtained for the first time from the genus Smenospongia. The structures of all compounds were established by spectroscopic methods (UV, IR, 1D and 2D NMR, MS, [alpha]D). The cytotoxic potential of 1-8 was evaluated in a panel of isogenic HCT-116 human colon tumor cell lines.     

N- and O-linked oligosaccharides of allergenic glycoproteins

Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing 1,3 fucose and 1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions.     

Characterisation of novel point mutations in the survival motor neuron gene SMN, in three patients with SMA

We report two novel mutations in three cases of spinal muscular atrophy (SMA), including two distant cousins who followed an unexpectedly severe course. Diagnosis was confirmed by reduced SMN protein and full-length SMN mRNA levels. Sequencing of the non-deleted SMN1 gene revealed a single G insertion at the end of exon 1 in the two cousins and a novel G275S exon 6 missense mutation in the milder case.     

Gonococcal MinD affects cell division in Neisseria gonorrhoeae and Escherichia coli and exhibits a novel self-interaction

The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae (MinD(Ng)) in this mutant. Hence, MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD(Ng) is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC(Ng) and MinD(Ng) from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC(Ng) or MinD(Ng) alone did not display noticeable morphological changes. These studies suggest that MinD(Ng) is involved in inhibiting gonococcal cell division, likely in conjunction with MinC(Ng). The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD(Ng) in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD(Ng) revealed a novel MinD(Ng) self-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinD(Ng). These results indicate that MinD(Ng) is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.