The ilvIH operon of Escherichia coli K-12. Identification of the gene products and recognition of the translational start by polypeptide microsequencing

The ilvI and ilvH gene products were identified physically by electrophoretic analysis of in vivo-labelled polypeptides produced in minicells from plasmids carrying the wild-type ilvIH operon of Escherichia coli K-12 and derivatives of it. An analysis of the distribution of methionine residues in the amino-terminal portion of micro-quantities of the ilvI product eluted from gel showed that the translational start of the ilvI gene is the promoter-proximal one of three putative methionine codons predicted from the DNA sequence.     

Early conception factor lateral flow assays for pregnancy in the mare

The ECF™ lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF™ test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.     

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.     

Adrenergic modulation of immune cells: an update

Sympathoadrenergic pathways are crucial to the communication between the nervous system and the immune system. The present review addresses emerging issues in the adrenergic modulation of immune cells, including: the specific pattern of adrenoceptor expression on immune cells and their role and changes upon cell differentiation and activation; the production and utilization of noradrenaline and adrenaline by immune cells themselves; the dysregulation of adrenergic immune mechanisms in disease and their potential as novel therapeutic targets. A wide array of sympathoadrenergic therapeutics is currently used for non-immune indications, and could represent an attractive source of non-conventional immunomodulating agents.     

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

Summary In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of ¹²⁵I-labelled human follicle-stimulating hormone (¹²⁵I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of ¹²⁵I-labelled human choriogonadotropin (¹²⁵I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.     

Substance P as a transglutaminase substrate: identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor, iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Sterols from Basidiomycetes

Sterols of three Basidiomycetes were determined. The main sterol was ergosta-5,7,22-trien-3 beta-ol, accompanied by other closely related sterols. Cholesterol was found only in trace amounts.