Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.     

Vanadyl tetradentate Schiff base complexes as catalyst for C-H bond activation of olefins with tert-butylhydroperoxide: Synthesis, characterization and structure

Three N₂O₂ tetradentate Schiff base ligands (H₂L^1-3) were prepared by reaction of 1,2-propylenediamine and appropriate aldehyde and ketone and characterized by FT-IR, ¹H and ¹³C NMR. The vanadyl complexes were synthesized by treating an ethanolic solution of the appropriate ligand and one equivalent of VO(acac)₂ to yield VOL^1-3. These oxovanadium (IV) complexes were characterized on the basis of their FT-IR, UV-Vis spectroscopy and elemental analysis. The crystal structure of VOL³ has been determined. The metal center in VOL³ is a deformed tetragonal pyramidal N₂O₃ coordination sphere. These complexes are used as catalyst for the selective epoxidation of olefins. High selectivity of epoxidation for cyclooctene observed from oxidation data. The catalytic activity increases as the number of electron-donor groups increases, and the catalytic selectivity is varied by changing the substituents on the ligands. The catalytic system described here is an efficient and inexpensive method for the oxidation of olefins, with the advantages of high activity, selectivity, re-usability and short reaction time.     

A Homozygous Nonsense Mutation in the Human Desmocollin-3 (DSC3) Gene Underlies Hereditary Hypotrichosis and Recurrent Skin Vesicles

Desmosomes are the major players in epidermis and cardiac muscles and contribute to intercellular binding and maintenance of tissue integrity. Two important constituents of desmosomes are transmembrane cadherins named desmogleins and desmocollins. The critical role of these desmosomal proteins in epithelial integrity has been illustrated by their disruption in mouse models and human diseases. In the present study, we have investigated a large family from Afghanistan in which four individuals are affected with hereditary hypotrichosis and the appearance of recurrent skin vesicle formation. All four affected individuals showed sparse and fragile hair on scalp, as well as absent eyebrows and eyelashes. Vesicles filled with thin, watery fluid were observed on the affected individuals'' scalps and on most of the skin covering their bodies. A scalp-skin biopsy of an affected individual showed mild hair-follicle plugging. Candidate-gene-based homozygosity linkage mapping assigned the disease locus to 8.30 cM (8.51 Mbp) on chromosome 18q12.1. A maximum multipoint LOD score of 3.30 (θ = 0.00) was obtained at marker D18S877. Sequence analysis of four desmoglein and three desmocollin genes, contained within the linkage interval, revealed a homozygous nonsense mutation (c.2129T>G [p.Leu710X]) in exon-14 of the desmocollin-3 (DSC3) gene.     

Molecular investigation of mental retardation locus gene PRSS12 by linkage analysis

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.     

Phytochemical and toxicological investigations of crude methanolic extracts,subsequent fractions and crude saponins of Isodon rugosus

Background

Isodon rugosus is used traditionally in the management of hypertension, rheumatism, tooth-ache and pyrexia. Present study was arranged to investigate I. rugosus for phytoconstituents, phytotoxic and cytotoxic activities to explore its toxicological, pharmacological potentials and to rationalize its ethnomedicinal uses. Briefly, qualitative phytochemical analysis of the plant extracts were carried out for the existence of alkaloids, flavonoids, saponins, oils, glycosides, anthraquinones, terpenoids, sterols and tannins. Plant crude methanolic extract (Ir.Cr), its subsequent fractions; n-hexane (Ir.Hex), chloroform (Ir.Chf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq) and saponins (Ir.Sp) in different concentrations were tested for phytotoxic and cytotoxic activities using radish seeds and brine shrimps (Artemia salina) respectively. The phytotoxic activity was determined by percent root length inhibition (RLI) and percent seeds germination inhibition (SGI) while the cytotoxicity was obtained with percent lethality of the brine shrimps.

Results

Ir.Cr was tested positive for the presence of alkaloids, glycosides, flavonoids, oils, terpenoids, saponins, tannins and anthraquinones. Among different fractions Ir.Sp, Ir.Chf, Ir.EtAc, and Ir.Cr were most effective causing 93.55, 89.32, 81.32 and 58.68% inhibition of seeds in phytotoxicity assay, with IC₅₀ values of 0.1, 0.1, 0.1 and 52 μg/ml respectively. Similarly, among all the tested samples, Ir.Sp exhibited the highest phytotoxic effect causing 91.33% root length inhibition with IC₅₀ of 0.1 μg/ml. Ir.Sp and Ir.Chf were most effective against brine shrimps showing 92.23 and 76.67% lethality with LC₅₀ values of 10 and 12 μg/ml respectively.

Conclusions

It may be inferred from the current investigations that I. rugosus contains different secondary metabolites and is a potential source for the isolation of natural anticancer and herbicidal drug molecules. Different fractions exhibited phytotoxic and cytotoxic activities, thus providing pharmacological basis for ethnomedicinal uses of this plant.     

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (< 90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced > 80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (< 90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.     

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2 nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2 nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′ → 3′ toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.