Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.     

Mutational change of membrane architecture. Mutants of Escherichia coli K12 missing major proteins of the outer cell envelope membrane

Mutant of Escherichia coli have been analyzed which miss two of the major proteins of the outer cell envelope membrane. The two proteins I and II1, normally are present at high concentrations (about 10⁵ copies per cell).In such mutants, as compared with wild type, the phospholipid-to-protein ratio in the outer membrane has increased by a factor of 2.3 causing a considerable difference in density between wild type and mutant membranes. The concentrations of two other major components of the outer membrane, lipopolysaccharide and Braun's lipoprotein, did not change.The protein-deficient mutants do not exhibit gross functional defects in vitro. An increased sensitivity to EDTA and a slight such increase to dodecyl sulfate (but not to deoxycholate or Triton X-100) was observed, loss of so-called periplasmic enzymes was not found, and other differences to wild type are marginal. The mutants can grow with normal morphology. It is not possible, however, to prepare “ghosts” (particles of size and shape of the cell without murein, surrounded by a derivative of the outer membrane, and posssessing the major proteins of this membrane) from them. This fact confirms our earlier suggestion that the proteins in question are required for the shape maintenance phenomenon in ghosts, and the mutants reject the speculation that these proteins are involved in the expression of the genetic information specifying cellular shape.Freeze-fracturing showed that in mutant cells, and in sharp contrast to wild type, the far predominant fracture plane is within the outer membrane. The concentration of the well known densely packed particles at the outer, concave leaflet of this fracture plane is greatly reduced. It was not possible, however, to clearly establish that one or the other protein is part of these particles because these ultrastructural differences were not apparent in mutants missing either one of the proteins only. The biochemical and ultrastructural data allow the conclusion that the loss of two major proteins and the concomitant increase of phospholipid concentration has changed the architecture of the outer membrane from a highly oriented structure. with a large fraction of protein-protein interaction, to one predominantly exhibiting planar lipid bilayer characteristics. E. coli thus can assemble rather different outer membranes, afact excluding that outer membrane formatin constitutes a highly ordered or strictly sequential assembly-line process.     

Postparturitional testosterone surge in male offspring of rats stressed and/or fed ethanol during late pregnancy

Male offspring of rats exposed to restraint stress and/or alcohol during late pregnancy show aberrant patterns of sexual behavior masculinization and defeminization that vary as a function of treatment. The impact of these treatments on the postparturitional testosterone (T) surge that contributes to sexual behavior differentiation was investigated. Plasma T was measured using radioimmunoassay in individual males sampled on day 21 of gestation within 10 min of cesarean delivery or 1, 2, or 4 h thereafter. Neonatal T in the group exposed only to stress did not differ from that in the control group. T was lower than control levels at birth in both alcohol groups. The magnitude of the T surge that occurred during the first hour of birth in the control group was diminished by 50% in both alcohol groups, whose T pattern was very similar. There was no common alteration in postparturitional T associated with the increased lordotic behavior potential that males in all three treatment groups typically share, nor were there idiosyncratic endocrine abnormalities linked to the very different male copulatory pattern each exhibits. Exposure to an abnormal T milieu during fetal as well as neonatal ontogeny may underlie the etiology of the different sexual behavior patterns exhibited by males exposed to stress and/or alcohol. Possible unique effects each treatment exerts on perinatal plasma T and it's aromatization to estradiol in hypothalamic targets are discussed.     

Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes

Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.     

The Influence of Nitrogen Nutrition on the Accumulation of Free Amino Acids in Root Tissue of Urtica dioica and their Apical Transport in Xylem Sap

Urtica dioica plants were grown on a nitrogen supply of 3, 15and 22 mM with nitrate and ammonium as nitrogen source. In contrastto nitrate reductions amino acid synthesis occurred in roottissue. At 3 mM ammonium obviously the amino acids were rathertransported via xylem upwards to the shoots than stored in theroots. Particularly increased ammonium supply led to stimulatedstorage of free amino acids in the roots, mainly as asparagineand arginine. In xylem asparagine was the dominant nitrogentransporting compound, while arginine was hardly translocated.With the enhancement of nitrogen supply, the second amide, glutamine,became more and more important with respect to the transportof nitrogen. (Received September 3, 1984; Accepted November 2, 1984)     

Monoclonal antibodies directed against rat liver epithelial cell lines selectively recognize bile duct epithelium in livers of adult rats

Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 40 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.Abbreviations ABTS2 2,2azinobis(3-ethylbenzthiazolinesulfonic) acid - ARL adult rat liver - DEN diethylnitrosamine - FCS fetal calf serum - MAb monoclonal antibody - PAP peroxidase antiperoxidase     

Synthesis and structure of 1,5-dideoxy-1,5-dihalogeno- and 1,2:4,5-dianhydro-xylitol derivatives

Eleven terminally disubstituted dihalogeno and diepoxy derivatives of xylitol have been prepared. Their structures were determined by ¹H-n.m.r. spectroscopy and, in one case, by X-ray diffraction.     

Electron microscopic and histochemical investigations of the atretic oocyte of Perca fluviatilis L. (Teleostei)

Summary The regression of atretic oocytes in Perca fluviatilis was studied by histochemical, light and electron microscopic methods. The course of regression can be divided into three stages, the first two comprising the dissolution of the atretic oocyte and its phagocytosis by the granulosa cells of the follicular epithelium, and the third stage consisting of the dissolution of the granulosa cells themselves. The ultrastructure in all three stages shows only features related to phagocytosis and lysosome formation. In particular, there is no agranular endoplasmic reticulum formed within the phagocytically active granulosa cells, nor is there any 3-hydroxysteroid dehydrogenase activity (3-HSD). Large yellow-orange pigments, formed during the third stage of regression, are ascribed to a relative deficiency of lysosomes in lipid digestion, and do not result from a preceeding steroid-synthesising phase as in mammalian corpora lutea. Thus, the atretic oocyte of P. fluviatilis is considered not to give rise to a corpus luteum formation with endocrine function, but merely represents a degenerative structure.