Synthesis and in vitro anti Mycobacterium tuberculosis activity of a series of phthalimide derivatives

New phthalimide derivatives were easily prepared through condensation of phthalic anhydride and selected amines with variable yields (70–90%). All compounds (3a–l) were evaluated against Mycobacterium tuberculosis H₃₇Rv using Alamar Blue susceptibility. The compounds 3c, 3i, and 3l have the minimum inhibitory concentrations (MICs) of 3.9, 7.8, and 5.0 μg/mL, respectively, and could be considered new lead compounds in the treatment of tuberculosis and multi-drug resistant tuberculosis.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.     

Mutagenicity,sister chromatid exchange inducibility andin vitro cell transforming ability of particulates from Athens air

Airborne particulates were collected over a period of twelve months by the use of Hi-Vol samplers in the basin of Athens, Greece. N-Hexane extracts were tested in a battery ofin vitro tests for their ability to induce mutation in bacteria as well as mutation, sister chromatid exchange and morphological transformation in cultured mammalian cells. Positive results were found for mutagenicity withSalmonella strain TA98 in the Ames assay, for sister chromatid exchange induction in CHO cells and for transformation in BALB/c 3T3 cells in culture. They also showed weak non-doserelated induction of ouabain resistance in BALB/c 3T3 cells. The contribution of oxidizing and nitrating agents found in the Athens atmosphere, together with sunlight UV irradiation in the formation of direct acting mutagens and potential carcinogens from ambient polycyclic aromatic hydrocarbons, is suggested.Abbreviations FCS fetal calf serum - FPG fluorescent-plus-Giemsa technique - oua^R ouabain resistant - PAH polycyclic aromatic hydrocarbon - SCE sister chromatid exchange - TSP total suspended particulate     

Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130

Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R), the cDNA of which has already been cloned. We report here that an 80 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37 degrees C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0 degree C. Human IL-6-R can associate with a murine gp130 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.