Hazir Rahman

Mycophenolic acid mediated disruption of the intestinal epithelial tight junctions

Gastrointestinal toxicity is a common adverse effect of mycophenolic acid (MPA) treatment in organ transplant patients, through poorly understood mechanisms. Phosphorylation of myosin light chain 2 (MLC2) is associated with epithelial tight junction (TJ) modulation which leads to defective epithelial barrier function, and has been implicated in GI diseases. The aim of this study was to investigate whether MPA could induce epithelial barrier permeability via MLC2 regulation. Caco-2 monolayers were exposed to therapeutic concentrations of MPA, and MLC2 and myosin light chain kinase (MLCK) expression were analyzed using PCR and immunoblotting. Epithelial cell permeability was assessed by measuring transepithelial resistance (TER) and the flux of paracellular permeability marker FITC-dextran across the epithelial monolayers. MPA increased the expression of MLC2 and MLCK at both the transcriptional and translational levels. In addition, the amount of phosphorylated MLC2 was increased after MPA treatment. Confocal immunofluorescence analysis showed redistribution of TJ proteins (ZO-1 and occludin) after MPA treatment. This MPA mediated TJ disruption was not due to apoptosis or cell death. Additionally ML-7, a specific inhibitor of MLCK was able to reverse both the MPA mediated decrease in TER and the increase in FITC-dextran influx, suggesting a modulating role of MPA on epithelial barrier permeability via MLCK activity. These results suggest that MPA induced alterations in MLC2 phosphorylation and may have a role in the patho-physiology of intestinal epithelial barrier disruption and may be responsible for the adverse effects (GI toxicity) of MPA on the intestine.

ANTIBACTERIAL ACTIVITY OF SOME MEDICINAL PLANTS AGAINST SELECTED HUMAN PATHOGENIC BACTERIA

Medicinal plants are traditionally used for the treatment of human infections. The present study was undertaken to investigate Bergenia ciliata, Jasminum officinale, and Santalum album for their potential activity against human bacterial pathogens. B. ciliata, J. officinale, and S. album extracts were prepared in cold and hot water. The activity of plant extracts and selected antibiotics was evaluated against five bacterial pathogens including Staphylococcus aureus, Bacillus subtilis, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli using agar well diffusion method. Among the three medicinal plants, B. ciliata extracts displayed potential activity against bacterial pathogens. Cold water extract of Bergenia ciliate showed the highest activity against B. subtilis, which is comparable with a zone of inhibition exhibited by ceftriaxone and erythromycin. J. officinale and S. album extracts demonstrated variable antibacterial activity. Further studies are needed to explore the novel antibacterial bioactive molecules.

Differential proteome analysis of human embryonic kidney cell line (HEK-293) following mycophenolic acid treatment

BackgroundMycophenolic acid (MPA) is widely used as a post transplantation medicine to prevent acute organ rejection. In the present study we used proteomics approach to identify proteome alterations in human embryonic kidney cells (HEK-293) after treatment with therapeutic dose of MPA. Following 72 hours MPA treatment, total protein lysates were prepared, resolved by two dimensional gel electrophoresis and differentially expressed proteins were identified by QTOF-MS/MS analysis. Expressional regulations of selected proteins were further validated by real time PCR and Western blotting.ResultsThe proliferation assay demonstrated that therapeutic MPA concentration causes a dose dependent inhibition of HEK-293 cell proliferation. A significant apoptosis was observed after MPA treatment, as revealed by caspase 3 activity. Proteome analysis showed a total of 12 protein spots exhibiting differential expression after incubation with MPA, of which 7 proteins (complement component 1 Q subcomponent-binding protein, electron transfer flavoprotein subunit beta, cytochrome b-c1 complex subunit, peroxiredoxin 1, thioredoxin domain-containing protein 12, myosin regulatory light chain 2, and profilin 1) showed significant increase in their expression. The expression of 5 proteins (protein SET, stathmin, 40S ribosomal protein S12, histone H2B type 1 A, and histone H2B type 1-C/E/F/G/I) were down-regulated. MPA mainly altered the proteins associated with the cytoskeleton (26%), chromatin structure/dynamics (17%) and energy production/conversion (17%). Both real time PCR and Western blotting confirmed the regulation of myosin regulatory light chain 2 and peroxiredoxin 1 by MPA treatment. Furthermore, HT-29 cells treated with MPA and total kidney cell lysate from MMF treated rats showed similar increased expression of myosin regulatory light chain 2.ConclusionThe emerging use of MPA in diverse pathophysiological conditions demands in-depth studies to understand molecular basis of its therapeutic response. The present study identifies the myosin regulatory light chain 2 and peroxiredoxin 1 along with 10 other proteins showing significant regulation by MPA. Further characterization of these proteins may help to understand the diverse cellular effects of MPA in addition to its immunosuppressive activity.

Alleviation of heavy metal toxicity and phytostimulation of Brassica campestris L. by endophytic Mucor sp. MHR-7

Heavy metal (HM) pollution is of great concern in countries like Pakistan where a huge proportion of human population is exposed to it. These toxic metals are making their way from water bodies to soil where it not only interferes with plant growth and development but also initiates serious health issues in human consuming the produce of such soils. Bioremediation is one of the most viable and efficient solution for the problem. Purpose of the current study was to isolate endophytic fungi from plants grown on HM contaminated soil and screen them for their ability to tolerate multiple HM including chromium (Cr6+), manganese (Mn2+), cobalt (Co2+), copper (Cu2+) and zinc (Zn2+). Out of 27 isolated endophytes, only one strain (MHR-7) was selected for multiple heavy metals tolerance. The strain was identified as Mucor sp. by 18S and 28S ribosomal RNA internal transcribed spacer (ITS) 1 and 4 sequence homology. The strain effectively tolerated up to 900µgmL-1 of these heavy metals showing no remarkable effect on its growth. The adverse effect of the heavy metals, measured as reduction of the fungal growth increased with increasing concentration of the metals. The strain was able to remove 60-87% of heavy metals from broth culture when supplied with 300µgmL-1 of these metals. A trend of decline in bioremediation potential of the strain was observed with increasing amount of metals. The strain removed metals by biotransformation and/or accumulation of heavy metal in its hyphae. Application of Mucor sp. MHR-7 locked down HM in tis mycelium thereby making them less available to plant root reducing HM uptake and toxicity in mustard. Besides its bioremediation potential, the strain was also able to produce IAA, ACC deaminase and solubilize phosphate making it excellent phytostimulant fungus. It is concluded that MHR-7 is an excellent candidate for use as biofertilizer in fields affected with heavy metals.

Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrug-resistant Mycobacterium tuberculosis isolates from Pakistan.

The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.

Toxic Metal Pollution in Pakistan and Its Possible Risks to Public Health.

Environmental pollution has increased many folds in recent years and in some places has reached levels that are toxic to living things. Among pollutant types, toxic heavy metals and metalloids are among the chemicals that pose the highest threat to biological systems (Jjemba 2004). Unlike organic pollutants, which are biodegradable, heavy metals are not degraded into less hazardous end products (Gupta et al. 2001). Low concentrations of some heavy metals are essential for life, but some of them like Hg, As, Pb and Cd are biologically non-essential and very toxic to living organisms. Even the essential metals may become toxic if they are present at a concentration above the permissible level (Puttaiah and Kiran 2008). For example, exposure to Zn and Fe oxides produce gastric disorder and vomiting, irritation of the skin and mucous membranes. Intake of Ni, Cr, Pb, Cd and Cu causes heart problems, leukemia and cancer, while Co and Mg can cause anemia and hypertension (Drasch et al. 2006). Similarly, various studies indicated that overexposure to heavy metals in air can cause cardiovascular disorders (Miller et al. 2007; Schwartz 2001), asthma (Wiwatanadate and Liwsrisakun 2011), bronchitis/emphysema (Pope 2000), and other respiratory diseases (Dominici et al. 2006).

Malaria burden in human population of Quetta, Pakistan.

Malaria is a serious global health challenge, which is responsible for more than one million deaths a year. Malarial infection is more prevalent in developing countries including Pakistan. Significant efforts have been made to control malaria; however, due to socio-environmental factors, it remains a frequent problem in Quetta. The present study was undertaken to determine the malarial incidence, species prevalence, and its demographic evaluation in human population of Quetta, Pakistan. A total of 1831 subjects, comprising 1072 male and 759 female presenting symptoms of malaria, were included in this study. Blood samples from clinically suspected individuals were subjected to the standard immunochromatographic and malaria parasite smear analysis for malaria diagnosis. Out of 1831 subjects, 338 (18.45%) patients were positive for malarial parasite while the species prevalence was found as 276 (81.66%) and 62 (18.34%) for Plasmodium vivax, and Plasmodium falciparum, respectively. Furthermore, seasonal variations gradual increase in the prevalence rate. The age group of 21-30 years (30.47%) was found more prone to malaria. The suspected malaria cases were found more frequent in rural (72.1%) as compared to urban (27.9%). In addition, the malaria burden was high in urban area (22.89%) population as compared to the rural area (16.74%) population. It was observed that the highest disease occurrence was caused by P. vivax, which reflects a serious threat for public health. The current findings will be helpful to plan effective strategies to prevent and control malaria in this area.

Chlorpyrifos and lambda cyhalothrin-induced oxidative stress in human erythrocytes: In vitro studies.

Pesticides are one of the most potentially harmful chemicals introduced into the environment, and their adverse impacts on non-target organisms can be significant. The present study was conducted to shed light on effects of locally used insecticides chlorpyrifos (CPF) and lambda cyhalothrin (LCT) on oxidative stress biomarkers in human erythrocytes. The activity of catalase (CAT), superoxide dismutase (SOD), and protein contents as well as the levels of malondialdehyde (MDA) and osmotic fragility (OF) were measured in human erythrocytes exposed to CPF at concentrations of 0, 100, 500, 1000, and 2000 ppm and LCT at concentrations of 0, 100, 300, 600, and 800 ppm for 1 h and 3 h at 37°C. MDA levels and OF of erythrocytes were significantly higher in erythrocytes incubated with CPF and LCT at increasing concentrations of both insecticides and increased incubation time. However, erythrocyte CAT and SOD activities were decreased at all concentrations of CPF and LCT tested. Protein oxidation products were decreased at lower doses of CPF (100 and 500 ppm); at higher doses (1000 and 2000 ppm), total protein content was increased compared with control. In contrast LCT was associated with decreased in protein contents at all the concentrations. These results clearly demonstrated that CPF and LCT can induce oxidative stress in human erythrocytes (in vitro).

Effect of IAA on in vitro growth and colonization of Nostoc in plant roots.

Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg-1ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity.

Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome

BackgroundThe effects of fetal calf serum (FCS) heat inactivation and bacterial lipopolysaccharide (LPS) contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM) cells were grown in FCS, either non-heated, or heat inactivated, having low (< 1 EU/mL) or regular (< 30 EU/mL) LPS concentrations. Protein lysates were resolved by 2-DE followed by phospho-specific and silver nitrate staining. Differentially regulated spots were identified by nano LC ESI Q-TOF MS/MS analysis.ResultsA total of four proteins (EIF3M, PRS7, PSB4, and SNAPA) were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE) as compared to cells grown in media with non-heated FCS (NHE). Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN) displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1).ConclusionThe present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.