Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Sensory receptors of the ovipositor ofTrichogramma maidis [Hym.:Trichogrammatidae]

Sensory receptors of the ovipositor ofTrichogramma maidis are described. Sense organs are found on the 2^nd valvifers (1 type), on the tip of the 3^rd valvulae (2 types) and on the 1^st valvulae (4 types). The nature and possible functions of these sensilla are discussed.      

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work     

Core temperature variability in diving king penguins (Aptenodytes patagonicus): a preliminary analysis

Core temperature was determined in two king penguins living in the wild at Ile de la Possession, Crozel Archipelago, using implantable four-channel temperature loggers. Core temperatures derived from bird no. 1 (sensor placed under the sternum, in the vicinity of the liver and upper stomach) were closely correlated with diving activity (as determined by an external light recorder), and ranged from 38.3°C, (on land) to a minimum of 37.2°C during a dive. Core temperatures measured in bird no. 2 showed that temperatures near the heart were generally 1°C lower than those under the sternum or in the lower abdomen. Core temperatures declined continuously during dives (by 0.8, 1.2 and 2.7°C in the lower abdomen, under the sternum and near the heart, respectively) and showed precipitous drops to 35°C, probably associated with ingestion of food. Temperatures measured near the heart fluctuated over a period of 288 s, corresponding to the duration (from the literature) of the surface/dive cycle. The relevance of these findings with respect to diving physiology, blood perfusion of tissues, tissue metabolism and aerobic dive limits is discussed.     

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

A cellular model for drug interactions on hematopoiesis: The use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis

A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3-azido-3-deoxythymidine, acetylsalicyclic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC₅₀) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.Abbreviations ARA-CC cytosine arabinoside - AS acetylsalicylic acid - AZTT 3-azido-3-deoxythymidine - BFUU burst-forming units - BM bone marrow - CFU colony-forming units - DOXX doxorubicin - FU 5-fluorouracil - glyAA glyAcophorin A - HCB human umbilical cord blood - IU international units - PCMEM human placenta-conditioned medium - VA sodium valproate