Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.     

Elemental composition of seston and nutrient dynamics in the Sea of Marmara

The Sea of Marmara, an intercontinental basin with shallow and narrowstraits, connects the Black and Mediterranean Seas. Data obtained during1991–1996 have permitted the determination of the elementalcomposition of seston in the euphotic zone and the N:P ratio of thesubhalocline waters of the Marmara Sea. Since primary production is alwayslimited to the less saline upper layer (15–20 m), of the Marmara Sea,the subhalocline waters of Mediteranean origin are always rich in nutrients(NO₃ + NO₂ = 8–10 μm, PO₄ = 0.8–1.2 μm) but depleted in dissolvedoxygen (30–50 μm) throughout the basin, yielding an -O_{_2} : N : P ratio of 178 : 9 : 1. Pollution of the surfacewaters since the 60s has modified the subhalocline nutrient chemistryslightly. In the euphotic zone, the N : P ratio of the seston changes from5.9 to 9.5 between the less and more productive periods. Though the biologyof the Marmara has changed significantly during the previous two decades,the close relationship observed between the elemental composition of thesurface seston and the NO₃ : PO₄ ratio of thesubhalocline waters strongly suggests that during the whole year primaryproduction throughout the basin and POM export to the lower layer remainnitrogen-limited. This suggestion needs to be confirmed by bio-assays,biological studies and sediment trap data from the upper subhaloclinedepths. Nonetheless, the counterflows in the Marmara basin possessrelatively low N : P ratios in both dissolved and particulate nutrients andextend as far as the adjacent seas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II

Abstract The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli , the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tet^tr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb -specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Differentiation of cells producing polypeptide hormones (ACTH,MSH, LPH, α- and β-endorphin,GH and PRL) in the fetal porcine anterior pituitary

Summary The aim of the present study on the fetal porcine pituitary was (1) to detect by means of the immunoperoxidase technique the earliest stages of cells producing polypeptide hormones: -MSH, ACTH, -LPH, - and -endorphin, growth hormone (GH) and prolactin (PRL), (2) to study the development of the synthesis and the storage of these hormones during fetal life, and (3) to detect whether several hormones can be located in one and the same cell.The corticotropic cells were revealed as the earliest functional elements of the fetal anterior pituitary. Our results indicate clearly that ACTH, -MSH, -LPH, - and -endorphin appear at 34 days in the same regular, round or ovoid cells; no differences in the time of their appearance could be observed. The ACTH-cells, irregular or angular in shape and endowed with cytoplasmic processes such as described in the adult pituitary, were not seen until day 50. The first GH-cells were detected between 40 to 45 days of fetal life. From day 45 to 90, the GH-cells greatly increased in number and in staining intensity of their progressively extending cytoplasmic area, but they displayed the same regular and round shape. The PRL-cells were the last cell type to appear in the fetal pituitary. The first PRL-cells, small in size and round or ovoid in shape with a high nucleus/cytoplasm ratio, were detected at day 70. At day 80, the PRL-cells increased in size and staining intensity. They displayed an irregular elongated or stellated shape and cytoplasmic processes resembling those characteristic of the adult pituitary. These data suggest that in the fetal porcine pituitary: (1) ACTH, -LPH and related peptides are synthesized and stored in the same cells, and (2) PRL and GH appear in individual cellular elements.