Tetrahydrobiopterin improves vascular endothelial function in ovariectomized rats

The goal of the present study is to investigate the role of tetrahydrobiopterin (BH4) in the vascular response in ovariectomized rats. Rats were randomly assigned to two groups: (1) sham group: sham-operated female rats, and (2) Ovx group: rats were ovariectomized. Our results have shown that the plasma 17 beta-estradiol levels in the Ovx group at the end of the experiment were significantly lower than in the sham group. Vasoreactivity assessed with intact aortic rings indicated that the phenylephrine-induced vasocontractile response to aortic rings from the Ovx group was greater than that of the sham group. In contrast, the vasodilator responses to acetylcholine and L-arginine (L-Arg) in the sham group were significantly greater than in the Ovx group. Differences in vasoreactivity in denuded aorta between the two groups were not noted. Moreover, exogenous BH4 significantly restored L-Arg-induced vasodilator responses in the Ovx group. However, this improvement effect was not found in the sham group. In addition, there were significant increases in superoxide anion production in aortic tissue and significant decreases in plasma nitric oxide levels in the Ovx group. Furthermore, BH4 contents in the aorta in the Ovx group were significantly decreased compared with the sham group. In conclusion, the present study demonstrates that the impairment of vascular reactivity was found in the ovariectomized rats. The possible mechanism of this defect may have resulted from the deficiency of available BH4. Thus, this study may provide a novel therapeutic strategy for the treatment of postmenopausal cardiovascular disorders.     

Seafood Allergy and Allergens: A Review

Seafoods are composed of diverse sea organisms and humans are allergic to many of them. Tropomyosin is a major allergen in many shellfish, especially crustacea and mollusks. Interestingly, tropomyosin has also been identified as an important allergen in other invertebrates including dust mites and cockroaches, and it has been proposed by some to be an invertebrate pan allergen. Different regions of shrimp tropomyosin bind IgE; 5 major IgE-binding regions have been identified in shrimp tropomyosin containing 8 epitopes. Mutations of these shrimp allergenic epitopes can reduce seafood allergenicity; methods utilizing such mutations will provide safer vaccines for more effective treatment of seafood-allergic patients, and in the future less-allergenic seafood products for consumption. Current address: (R. Ayuso) St. John's Episcopal Hospital, South Shore 327 Beach 19th St., Far Rockaway, NY 11691, U.S.A. Current address: (G. Reese) Paul-Ehrlich-Institut, Department of Allergology, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany     

Patterns of protein carbonylation following oxidative stress in wild-type and<Emphasis Type="Italic"> sigB Bacillus subtilis</Emphasis> cells

Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the ^B-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the ^B-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and sigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by ^B-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.Communicated by W. Goebel     

Does the size of small objects influence chemical reactivity in living systems?

Previous theoretical works showed that chemical reactions in micro- and nano-droplets, bubbles and solid particles were strongly affected by their confinement. In particular, the smallness of the systems leads to high internal pressure compared to the external pressure, which then significantly modifies the values of chemical equilibrium and kinetic constants. In addition, surface tension or surface stress, reactional dilatation and surface charge play also a major role on the chemical reactivity. As living systems are also made of very complex dispersed subsystems, i.e. organelles, it seemed obvious to illustrate our theory by some biological actual examples encountered in pulmonary alveolae, in vacuolae and in medical applications, such as dissolution of gallstones.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.     

Kinetics of Ampicillin Synthesis Catalyzed by Penicillin Acylase from E. coli in Homogeneous and Heterogeneous Systems. Quantitative Characterization of Nucleophile Reactivity and Mathematical Modeling of the Process

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme–nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous (aqueous solution–precipitate) systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous aqueous solution–precipitate systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells?

H. Ohsaki, E. Hirakawa, Y. Kushida, S. Yokoshita, M. Nakamura, H. Kiyomoto and R. Haba Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells? Objective: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low‐grade urothelial carcinoma cells (LG‐UCs). Methods: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG‐UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette‐like arrangement, (iv) hobnail‐shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann–Whitney U‐test and chi‐square test, with differences considered significant at P < 0.05. Results: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG‐UCs. In LG‐UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. Conclusions: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG‐UCs.