Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Pigment destruction by Calanus pacificus: impact on the estimation of water column fluxes

Measurements of gut pigment made prior to fecal pellet productionin Calanus pacificus females and CV copepodites suggest that(i) chlorophyll a and/or its pheopigment derivatives are degradedinto molecules that are not detectable by the standard fluorometrictechnique; and (ii) the percentage of ingested chlorophyll awhich degrades into fluorometrically undetected molecules isnot constant. Thus, measurements of chlorophyll and pheopigmenta in the guts of zooplankton can only yield minimum estimatesof in situ grazing rates. Estimates of the vertical flux ofprimary particulates based on chlorophyll and pheopigment abudgets may also be underestimated.     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

Human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cystine

The state of the five half-cystine residues in human tissue factor (TF) has been characterized. The results indicate that the four half-cystines in the extracellular domain of TF form two disulfide bonds and the half-cystine in the cytoplasmic region is acylated by palmitic acid and stearic acid. The extracellular disulfide cross-links, Cys49-Cys57 and Cys186-Cys209, were deduced from the analysis of tryptic peptides. Acylation of the cytoplasmic half-cystine was demonstrated by purifying and characterizing fibroblast TF from cells labeled with [3H]palmitic acid. Radiolabeled fibroblast TF was observed by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tritiated material covalently bound to the protein was identified as [3H]palmitate and [3H]stearate by reverse-phase high-pressure liquid chromatography. Deacylation of TF with hydroxylamine resulted in the spontaneous generation of disulfide-linked TF dimers. This result suggests that the disulfide-linked TF dimer, a minor component of most TF preparations, and the recently described heterodimeric form of TF are artifacts produced by deacylation of Cys245 and subsequent interchain disulfide bond formation.     

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Activity and regulation of an amidase (acylamide amidohydrolase,EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

The Brevibacterium R 312 strain has an amidase with a wide substrate spectrum previously named acetamidase. The study of its activity showed that this enzyme was able to hydrolyze a large number of amides into their corresponding organic acids. The affinity of this enzyme for the substrates varied according to the length of the carbon chain and the spatial crowding of the molecule. The comparison of the specific rates of hydrolysis showed that propionamide was the amide substrate most quickly hydrolyzed.We confirmed the inducible feature of this enzyme and noted that only acetamide and N-methylacetamide were inducers of this enzyme among the compounds tested. Thioacetamide and N-methylpropionamide, both as amide analogues, were shown to inhibit the biosynthesis of acetamidase. Similarly, the organic acids, products of the hydrolysis reaction, showed a strong repression action on the biosynthesis of the enzyme.     

New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics

Cryoenzymology was initially used to slow down enzyme-catalyzed reactions so as to stabilize intermediates for further study. During the course of this early work, it became clear that cryoenzymology could be extended to other ends and some of these are described. First, the use of a cryosolvent on its own (or together with temperature) as a perturbant has allowed a resolution of the substrate binding steps of certain enzymes (myosin, D-amino acid oxidase, peroxidase and cytochrome P450). Second, by the use of cryosolvent and temperature, coupled with the classical physico-chemical perturbants, one can selectively modulate the various steps of an enzyme pathway. This approach can lead to an understanding of the mechanism of enzyme regulation. Finally, by carrying out experiments over a wide range of temperatures (-30 degrees C- +30 degrees C) and pressure (up to several kbars) in specially constructed fast reaction equipment, one can study the thermodynamic properties of the individual rate constants describing the interconversions of reaction intermediates. Experiments with creatine kinase, cytochrome P450 and peroxidase are described. The thermodynamic parameters delta H, delta G, delta S and delta V are thus measured and when this is done under different solvent conditions one can, at least within the theories available, attempt an approach to the problem of protein dynamics.     

Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with Salmonella spp., and more than 90% of all food sources contained Escherichia coli. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of Salmonella isolates and 83.8% of E. coli isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of E. coli and 13% of Salmonella isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including aadA1, aadA2, and aadA5 (resistance to streptomycin and spectinomycin), aacA4 (resistance to aminoglycosides), the dihydrofolate reductase gene cassettes dhfrXII, dfrA1, and dhfrA17 (trimethoprim resistance), the beta-lactamase gene bla_PSE1 (ampicillin resistance), and catB3 (chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant Salmonella isolates and in 33 E. coli isolates. Thirty-five percent of the Salmonella isolates and 76% of the E. coli isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the Salmonella and E. coli food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria.     

MicroRNA-21 as a predictor and prognostic factor for trastuzumab therapy in human epidermal growth factor receptor 2-positive metastatic breast cancer

Breast cancer is the second most common cancer diagnosed worldwide. Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents about 20% to 30% of all breast cancers. Trastuzumab is used in the treatment of HER2-positive breast cancer. MicroRNA-21 (miR-21) is an oncomiR that acts by inhibiting many tumor-suppressor genes. We analyzed the relative expression levels of serum miR-21 in 20 HER2-positive metastatic breast cancer patients before and after 3 months of treatment with trastuzumab. miR-21 levels decreased with a high significant difference after trastuzumab therapy (P = 0.001). Although miR-21 expression levels were lower in responders than in nonresponders, the difference was not statistically significant ( P = 0.6). Our results demonstrated a significant negative correlation between its basal expression, expression levels after treatment, and time to progression ( P = 0.03 and 0.01, respectively). These results make miR-21 a potential prognostic factor for HER2-positive metastatic breast cancer patients. Additionally, it can be an interesting potential target in therapy using antisense oligonucleotides for miR-21.