Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

Summary We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

Kidney atrophy vs hypertrophy in diabetes: which cells are involved?

One of the first structural changes in diabetic nephropathy (DN) is the renal enlargement. These changes resulted in renal hypertrophy in both glomerular and tubular cells. Shrink in the kidney size, which described as kidney atrophy resulted from the loss of nephrons or abnormal nephron function and lead to loss of the kidney function. On the other hand, increase in kidney size, which described as hypertrophy resulted from increase in proximal tubular epithelial and glomerular cells size. However overtime, tubular atrophy and tubulointerstitial fibrosis occurs as subsequent changes in tubular cell hypertrophy, which is associated with the infiltration of fibroblast cells into the tubulointerstitial space. The rate of deterioration of kidney function shows a strong correlation with the degree of tubulointerstitial fibrosis. A consequence of long-standing diabetes/hyperglycemia may lead to major changes in renal structure that occur but not specific only to nephropathy. Identifying type of cells that involves in renal atrophy and hypertrophy may help to find a therapeutic target to treat diabetic nephropathy. In summary, the early changes in diabetic kidney are mainly includes the increase in tubular basement membrane thickening which lead to renal hypertrophy. On the other hand, only renal tubule is subjected to apoptosis, which is one of the characteristic morphologic changes in diabetic kidney to form tubular atrophy at the late stage of diabetes.     

Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia

We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G. orientalis in northern regions so far.     

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.     

Retinol enhances differentiation of the gastric parietal cell lineage in developing rabbits.

In the gastric glands, parietal cells are the targets for anti-ulcer drugs because they contain the proton pump or HK-ATPase responsible for acid secretion. Little is known about factors influencing developmental expression and activity of HK-ATPase. In this study, the parietal cell lineage was investigated in rabbits at post-natal days 0 (P0) to P60 by using morphological and biochemical methods. Immunohistochemical and ultrastructural studies show that the HK-ATPase-expressing cells that appear at P0 and P3 are pre-parietal cells. However, terminally differentiated, mature parietal cells make their appearance at P7. These data correlate with the activity of HK-ATPase, measured as K(+)-dependent hydrolysis of p-nitrophenyl phosphate. Three-day-retinol treatment of P3-P30 rabbits induced an increase in the (i) production of parietal cells, (ii) intensity of the HK-ATPase immunostaining per cell, (iii) activity of HK-ATPase and (iv) amount of HK-ATPase protein measured by Western blotting. In conclusion, retinol upregulates the development of HK-ATPase in rabbits, perhaps due to precocious acceleration of the differentiation program of parietal cell lineage.     

Antimicrobial Activity of Scabiosa arenaria Forssk. Extracts and Pure Compounds Using Bioguided Fractionation

The emergence of multidrug resistant pathogens threatened the clinical efficacy of many existing antibiotics. This situation has been recognized globally as a serious concern and justifies further research to discover antimicrobial agents from natural origins including plant extracts. The aim of our work was to evaluate the antimicrobial activities of Scabiosa arenaria Forssk . extracts and pure compounds using a bioguided fractionation, and try to explain some traditional use of this genus. The best antimicrobial activity‐guided fractionation was obtained by BuOH fractions of flowers, fruits and (stems and leaves) against Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory concentration (MIC) values from 0.0195 to 5 mg/ml. Escherichia coli was the most affected bug, thus the MIC of fruits BuOH extract showed the best anti‐Escherichia coli activity (MIC = 0.0195 mg/ml), followed by the (stems and leaves) and flowers BuOH extracts; MIC = 0.078 and 0.15 mg/ml, respectively. Furthermore, the subfractions obtained from these three mixed fractions showed also an important antimicrobial activity against the three microorganisms, with MIC values between 0.0195 and 0.312 mg/ml. The fractionation of the aerial part BuOH fraction led to the isolation of oleanolic acid ( 1 ) and luteolin 7‐O‐glucopyranoside ( 2 ) which are reported here for the first time from S. arenaria. Both compounds showed good antimicrobial activities with MIC values ranging from 170 to 683 μm and 86 to 347 μm , respectively. These results support the use of the Scabiosa genus to inhibit the growth of tested pathogenic bacteria and yeasts which may reduce illnesses associated with their exposure.     

Cervical Spinal Cord Atrophy Profile in Adult SMN1-Linked SMA

PurposeThe mechanisms underlying the topography of motor deficits in spinal muscular atrophy (SMA) remain unknown. We investigated the profile of spinal cord atrophy (SCA) in SMN1-linked SMA, and its correlation with the topography of muscle weakness.ResultsCSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10⁻⁵). There were no correlations between atrophy measurements, strength and disability scores.ConclusionsSpinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in more complex mechanisms underlying weakness in these patients.     

A Critical Analysis of Concentration and Competition in the Indian Pharmaceutical Market

Objectives

It can be argued that with several players marketing a large number of brands, the pharmaceutical market in India is competitive. However, the pharmaceutical market should not be studied as a single market but, as a sum total of a large number of individual sub-markets. This paper examines the methodological issues with respect to defining the relevant market involved in studying concentration in the pharmaceutical market in India. Further, we have examined whether the Indian pharmaceutical market is competitive.

Methods

Indian pharmaceutical market was studied using PharmaTrac, the sales audit data from AIOCD-AWACS, that organises formulations into 5 levels of therapeutic classification based on the EphMRA system. The Herfindahl-Hirschman Index (HHI) was used as the indicator of market concentration. We calculated HHI for the entire pharmaceutical market studied as a single market as well as at the five different levels of therapeutic classification.

Results and Discussion

Whereas the entire pharmaceutical market taken together as a single market displayed low concentration (HHI = 226.63), it was observed that if each formulation is defined as an individual sub-market, about 69 percent of the total market in terms of market value displayed at least moderate concentration. Market should be defined taking into account the ease of substitutability. Since, patients cannot themselves substitute the formulation prescribed by the doctor with another formulation with the same indication and therapeutic effect, owing to information asymmetry, it is appropriate to study market concentration at the narrower levels of therapeutic classification.