Correlation between pyridoxal-5'-phosphate levels and the percentage activation of aspartate aminotransferase enzyme in haemolysate and plasma during in vitro incubation studies with different B6 vitamers

Conflicting results using erythrocyte aminotransferase (eAST) stimulation to assess vitamin B6 nutritional status in patients with less severe B6 deficiencies are common. It has been claimed that the presence of different B6 vitamers may modify the activation of eAST by pyridoxal-5'-phosphate (PLP) leading to stimulatory or even inhibitory effects. To investigate the possible role of this phenomenon in producing inconsistent AST stimulations, aliquots of whole blood were incubated with equivalent amounts of different B6 vitamers, and the AST stimulation was correlated with the concentrations of PLP, measured by high-performance liquid chromatography. At the end of the incubation period the erythrocytes and plasma were separately analyzed. The conversion of non-PLP B6 vitamers to PLP, by the erythrocytes, was similar (approximately 70%) for all B6 vitamers used in the incubation experiments. The newly formed PLP accumulated in the erythrocytes, but the percentage activation of AST did not change significantly from the basal levels, in spite of the presence of increased levels of PLP and other B6 vitamers used for incubation. When PLP was used in the incubation studies, all of it was retained by the plasma and was associated with a marked suppression of plasma AST stimulation. To determine the degree to which plasma and erythrocyte AST was dose-dependent, plasma and haemolysates were incubated with increasing concentrations of PLP. A very significant inverse relationship was obtained in plasma between AST stimulation and PLP even at modest PLP levels, while haemolysates required incubation with much higher PLP concentrations to demonstrate the same effect. Since plasma PLP is considered to be the most reliable indicator of B6 nutritional status in man, our findings suggest that plasma percentage AST stimulation more closely reflects the B6 nutritional status than erythrocyte AST stimulation test which may reflect B6 status only in severe, longstanding B6 deficiencies. Conflicting results using erythrocyte AST stimulations may be attributed to the insensitivity of red cell AST to changes in PLP content. It is unlikely that the presence of non-PLP B6 vitamers in haemolysate may affect the percentage stimulation of aminotransferase enzymes by PLP.     

Rotavirus-associated diarrhea in a commercial rabbitry

An epizootic of diarrheal disease occurred in a commercial specific-pathogen-free rabbitry, and was characterized by sudden onset, rapid spread, and high morbidity and mortality among sucklings. Affected rabbits rapidly became dehydrated and most died within two days of the onset of diarrhea. Eight of these rabbits were necropsied. Five had blunted and fused small intestinal villi with attenuated villous enterocytes. A rotavirus was isolated from four rabbits, and five survivors of affected litters had strong antibody responses to rotavirus.     

Inclusion body disease of falcons (Herpesvirus infection) in an American kestrel.

Postmortem examination of a captive-bred American kestrel (Falco sparverius) showed numerous white necrotic foci 1-2 mm in diameter throughout the liver and spleen. The results of light and spleen. The results of light and electron microscopic studies and experimental transmission to a captive American kestrel and a barred owl (Strix varia) suggests a herpesvirus similar to those dsecribed for owls and other falcons in the U.S. This is the first report of a naturally occurring case of inclusion body disease of falcons in the American kestrel.     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Steroidogenic correlates of pregnancy in the rock hyrax (Procavia capensis)

In pregnant rock hyraxes isolated leucocytes metabolise both [3H]pregnenolone and [3H]progesterone while whole blood, erythrocytes and an erythrocyte/leucocyte mixture only metabolised [3H]progesterone. Plasma displayed no tendency to metabolically convert any one of these two steroids. In whole blood [3H]progesterone appears to be converted to 5alpha-pregnane-3,20-dione and a compound with chromatographic properties similar to that of 5alpha-pregnan-3alpha-ol-20-one. 5Alpha-pregnane-3,20-dione exhibited a high relative binding affinity for the uterine progesterone eceptor (94%), but 5alpha-pregnan-3alpha-ol-20-one displayed very little affinity for the same receptor (0.4%). 5Alpha-pregnane-3,20-dione may therefore aid in the maintenance of pregnancy. Corpora lutea metabolised progesterone to 17alpha-hydroxyprogesterone, a compound exhibiting no progestational function because of its low relative binding affinity for the uterine progesterone receptor (2%). Progesterone appears to be the main product of the corpus luteum. However, 5alpha-pregnane-3,20-dione circulated at concentrations approximately 8.5 times higher than progesterone, probably due to the metabolic conversion of progesterone to 5alpha-pregnane-3,20-dione by the blood. We conclude that in the hyrax progesterone, produced by the corpora lutea, enters the circulation, where it is reduced to 5alpha-pregnanes. 5Alpha-pregane-3,20-dione may then be transported to the uterus where it binds to the progesterone receptor to assist in the maintenance of pregnancy. This mechanism appears to be analogous to that of the African elephant which is phylogenetically related to the hyrax, except that in the elephant the 5alpha-reduced metabolites are produced by luteal tissue and not the blood.     

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose

A significant percentage of eukaryotic proteins contain posttranslationalmodifications, including glycosylation, which are required forbiological function. However, the understanding of the structure–functionrelationships of N-glycans has lagged significantly due to themicroheterogeneity of glycosylation in mammalian produced proteins.Recently we reported on the cellular engineering of yeast toreplicate human N-glycosylation for the production of glycoproteins.Here we report the engineering of an artificial glycosylationpathway in Pichia pastoris blocked in dolichol oligosaccharideassembly. The PpALG3 gene encoding Dol-P-Man:Man₅GlcNAc₂-PP-Dolmannosyltransferase was deleted in a strain that was previouslyengineered to produce hybrid GlcNAcMan₅GlcNAc₂ human N-glycans.Employing this approach, combined with the use of combinatorialgenetic libraries, we engineered P. pastoris strains that synthesizecomplex GlcNAc₂Man₃GlcNAc₂ N-glycans with striking homogeneity.Furthermore, through expression of a Golgi-localized fusionprotein comprising UDP-glucose 4-epimerase and ß-1,4-galactosyltransferase activities we demonstrate that this structure isa substrate for highly efficient in vivo galactose addition.Taken together, these data demonstrate that the artificial invivo glycoengineering of yeast represents a major advance inthe production of glycoproteins and will emerge as a practicaltool to systematically elucidate the structure–functionrelationship of N-glycans. ¹ These authors contributed equally to this work. ² To whom correspondence should be addressed; e-mail: swildt{at}glycofi.com