Kinetic properties of Streptomyces canarius L- Glutaminase and its anticancer efficiency

Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).

Evaluation of Cellular Proliferative Activity in Patients with Oral Lichen Planus and Hepatitis C through AgNOR Method

The objective of this study was to evaluate the cellular proliferative potential of oral lichen planus (OLP) lesions from patients without hepatitis C virus (HCV) by means of AgNOR method, as well as the cellular proliferative potential of the normal oral mucosa from patients with HCV, treated or untreated by interferon and ribavirin. A cross-sectional study was developed to investigate four groups: 10 HCV+ patients without clinical signs of OLP who had never been treated for HCV infection - Group 1; 10 HCV+ patients that were under interferon and ribavirin treatment - Group 2; 15 patients with reticular OLP lesions histopathologically confirmed, without HCV - Group 3; and 15 blood donors without HCV infection and no clinical signs of OLP GROUP 4 Control Group. The cytological material of all groups was collected by the liquid-based cytology technique. Then, the sedimented material from each patient was filled with the Nucleolar Organizer Regions impregnation by silver method (AgNOR). The count of NORs was performed on 100 epithelial cell nuclei per patient using the Image Tool(tm) software. The Tukey HSD test was used to compare the median value of NORs among the groups and showed that the oral mucosa of HCV+ patients previously treated with anti-HCV drugs (GROUP 2), presented a higher average number of NORs in relation to others (p<0.05). The anti-HCV treatment may be related to increased cell proliferation of oral mucosa, indicating a possible relationship between OLP and HCV+ patients treated with interferon and ribavirin.

O propósito deste estudo foi avaliar o potencial proliferativo celular das lesões de líquen plano bucal (LPB) de pacientes sem vírus da hepatite C (VHC) por meio do método AgNOR, comparando-o ao potencial proliferativo celular da mucosa bucal normal de portadores de VHC, tratados ou não com interferon e ribavirina. Um estudo transversal foi realizado para investigar 4 grupos: 10 pacientes VHC+ sem sinais clínicos de LPB que nunca haviam sido tratados para a infecção por VHC - Grupo 1; 10 pacientes VHC+ que estavam sob tratamento com interferon e ribavirina - Grupo 2; 15 pacientes com LPB reticular histopatologicamente confirmado, sem VHC - Grupo 3; e 15 doadores de sangue sem infecção por VHC e sem sinais clínicos de LPB (Grupo 4 - Grupo de Controle). O material celular de todos os grupos foi coletado pela técnica da citologia em base líquida. Então, o material sedimentado de cada paciente foi submetido ao método da impregnação das regiões organizadoras nucleolares pela prata (AgNOR). A contagem das NORs foi realizada em 100 núcleos celulares epiteliais por paciente por meio do programa Image Tool(r). O teste Tukey HSD foi utilizado para comparar o valor médio de NORs entre os grupos e mostrou que a mucosa bucal dos pacientes VHC+ previamente tratados com fármacos anti-VHC (Grupo 2) apresentou maior número médio de NORs por núcleo em relação aos outros (p<0,05). O tratamento anti-VHC pode estar relacionado ao aumento da atividade proliferativa celular da mucosa bucal, aventando uma possível relação entre LPB e pacientes VHC+ tratados com interferon e ribavirina.

Rise in free intracellular calcium in HeLa cells infected with aggregative Klebsiella pneumoniae strains isolated from cases of diarrhoea.

BACKGROUND & OBJECTIVES: Klebsiella pneumoniae strains occasionally cause diarrhoea in humans. This study was done to determine the involvement of calcium in the pathogenesis of aggregative K. pneumoniae strains. METHODS: A total of nine strains of K. pneumoniae were tested for adherence assay in HeLa cell line. A representative strain CO-1215 was used for [Ca2+]i study using Fura-2 fluorescence. RESULTS: Infection of cultured HeLa cells with aggregative K. pneumoniae strain resulted in five-fold elevation of intracellular level of free calcium ([Ca2+]i) with maximum Ca2+ influx at 3 h after bacterial infection. Chelation of extracellular Ca2+ with [ethylenebis(oxyethylenenitrile)] tetraacetic acid and suspension of cells in Ca2+ free buffer suggested that the rise of Ca2+ in aggregative K. pneumoniae infected HeLa cells was due to influx of Ca2+ from extracellular medium. INTERPRETATION & CONCLUSIONS: This study showed aggregative adherence in HeLa cells and this adherence leads to influx of extracellular Ca2+. The unrestricted passage of calcium ions across cell membranes could cause phosphorylation of proteins involved in ion transport across the membrane, which could result in secretory diarrhoea. Further work is in progress to study the enterotoxicity of these strains in an in vitro rabbit intestinal model.

Endoplasmic reticulum retention and degradation of T cell antigen receptor beta chain

The T cell antigen receptor-CD3 (TCR/CD3) complex is assembled in the endoplasmic reticulum (ER) of T cells after synthesis of individual chains, and is transported to the cell surface for immune recognition and regulation. Partially assembled or unassembled TCR chains are retained and rapidly degraded in the ER. These processes are strictly regulated in the ER at post-translational level for the maintenance of cellular homeostasis. In order to identify the region responsible for the ER retention and rapid degradation of the TCR beta chain, number of mutants were engineered and their fates, after synthesis in the ER of the HeLa cells, were investigated. Extensive mutagenic analysis of TCR beta chain, including changing the charged amino acid residues and two tyrosine residues of the transmembrane region into hydrophobic amino acid residues, did not alter the ER retention and rapid degradation. Soluble TCR beta chain and cytoplasmic tail truncation mutant were also rapidly degraded in the ER. However, N-glycosylation rate of soluble TCR beta chain in the ER was significantly increased possibly due to the increased exposure of the N-glycosylation site. These results suggest that the ER retention of TCR beta chain is mediated through its extracellular and transmembrane-cytoplasmic regions and that the rapid ER degradation can be caused by an exposure of unassembled subregion of TCR beta chain, either extracellular domain or hydrophobic transmembrane region to the hydrophilic environment (lumen of the ER) rather than by presence of a specific degradation signal.