Molecular characterization of a defensin in the IZD-MB-0503 cell line derived from immunocytes of the insect Mamestra brassicae (Lepidoptera)

The induction of anti-microbial peptides against Gram positive and negative bacteria in the IZD-MB-0503 cell line from the lepidopteran Mamestra brassicae is demonstrated, while no anti-fungal activity is detected. The identification of a defensin-like molecule active against Gram positive bacteria is described for the first time in Lepidoptera. This molecule shows between 43% and 59% homology with group A defensins from other dipteran and hymenopteran species.     

Dynamic,thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3'-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn(2+), the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide approximately 8 orders of magnitude in the affinity (Delta G degrees approximately equal to -10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to approximately 1.5 orders of magnitude (Delta G degrees approximately equal to - 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the k(cat) term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.     

Carbonic anhydrase inhibitors. inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with anions

Except for sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane, tumor-associated isozyme CA IX with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, etc., are also provided for comparison. Isozyme IX has an inhibition profile by anions different in some aspects from those of CA I and CA II, that may have interesting physiological consequences.     

Synthesis, radiosynthesis and in vivo evaluation of 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-[(11)C]one, as a potent NR(1A)/2B subtype selective NMDA PET radiotracer

Recently, a new series of potent and highly subtype-selective 1-(heteroarylalkynyl)-4-benzylpiperidine antagonists of the NMDA receptors has been described by Pfizer Laboratories. In this series, 5-[3-(4-benzylpiperidin-1-yl)prop-1-ynyl]-1,3-dihydrobenzoimidazol-2-one (1) was identified as a selective antagonist for the NR1(A)/2B subtype, displaying IC(50) values for inhibition of the NMDA responses of 5.3 nM for this subtype (compared to NR1(A)/2A: 35 microM and NR1(A)/2C>100 microM) and was active in rat at a relatively low dosage (10mg/kg po). Derivative 1 has been synthesized in four chemical steps in good overall yield and labelled with carbon-11 at its benzoimidazolone ring using [(11)C]phosgene. The pharmacological profile of [(11)C]-1 was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [(11)C]-1 was extremely low (0.07% I.D./mL on average at 30 min) and rather uniform across the different brain structures. This in vivo brain regional distribution of [(11)C]-1 did not match with autoradiographic or binding data obtained with other NR2B subtype-selective NMDA ligands. Competition studies with ifenprodil (20 mg/kg, ip, 30 min before the radiotracer injection) failed to demonstrate specific binding of the radiotracer in the brain. In view of these results, and especially considering the low brain penetration of the radiotracer, [(11)C]-1 does not have the required properties for imaging NMDA receptors using positron emission tomography.     

50 Hz magnetic fields of varying flux intensity affect cell shape changes in invertebrate immunocytes: the role of potassium ion channels

The effect induced by exposure to 50 Hz magnetic fields (MFs) in immunocytes from the mussel Mytilus galloprovincialis is evaluated. The whole animal was exposed for 15 and 30 min to MF intensities ranging from 200 to 1,000 microT. The changes in the cellular shape of immunocytes, expressed as shape factor (SF), were studied at different times after addition of the chemotacting substance N-formyl-Meth-Leu-Phe (fMLP). Results show that MFs provoke differing delays in fMLP-induced cellular shape changes: 200 microT are ineffective, while levels from 300 microT upwards cause a significant increase in immunocyte SF values compared to controls. Reactivation of the cells is possible up to an intensity of 600 microT. The use of PCO 400, an opener of ATP-sensitive K+ channels, shows that potassium channels are involved in the effect of MFs on M. galloprovincialis immunocytes.     

Hypergravity impairs angiogenic response of in vitro cultured human primary endothelial cells

A variety of evidence suggest that cardiovascular system functions are impaired in altered gravity conditions. In this study we investigated the influence of hypergravity environment (3g) on endothelial cell proliferation, endothelial vasoactive compound production and on in vitro angiogenesis. We found that cultured primary human endothelial cells were very sensitive to mild hypergravity conditions. Even if we did not record changes in cell viability and apoptosis, we found significant differences in cell proliferation, prostacyclin (PGI2) synthesis, nitric oxide (NO) synthesis, and in angiogenic responses. Using western blotting technique we detected an increased expression of cycloxygenase-2 (COX-2) in primary endothelial cells exposed for 48 hours to hypergravity, in comparison to those exposed to normal gravity.     

CL316,243 and cold stress induce heterogeneous expression of UCP1 mRNA and protein in rodent brown adipocytes.

Uncoupling protein 1 (UCP1), the mammalian thermogenic mitochondrial protein, is found only in brown adipocytes, but its expression by immunohistochemistry is not homogeneous. Here we present evidence that the non-homogeneous pattern of immunostaining for UCP1 (referred to as the "Harlequin phenomenon") is particularly evident after acute and chronic cold (4C) stimulus and after administration of a specific beta(3)-adrenoceptor agonist (CL316,243). Accordingly, mRNA in situ expression confirmed the UCP1 non-homogeneous pattern of gene activation under conditions of adrenergic stimulus. Furthermore, morphometric analysis of immunogold-stained thin sections showed that UCP1-gold particle density was different among neighboring brown adipocytes with mitochondria of the same size and density. When the adrenergic stimulus was reduced in warm-acclimated animals (28C), UCP1 protein and mRNA expression was reduced and consequently the Harlequin phenomenon was barely visible. These data suggest the existence of an alternative and controlled functional recruitment of brown adipocytes in acute adrenergically stressed animals, possibly to avoid heat and metabolic damage in thermogenically active cells. Of note, the heat shock protein heme oxygenase 1 (HO1) is heterogeneously expressed in adrenergically stimulated brown adipose tissue and, specifically, cells expressing strong immunoreactivity for UCP1 also strongly express HO1.     

MSJ-1, a New Member of the DNAJ Family of Proteins, Is a Male Germ Cell-Specific Gene Product

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein–protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages andin situhybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.