Coagulation proteases and neurotransmitters in pathogenicity of glioblastoma multiforme.

Glioblastoma is an aggressive type of cancer that begins in cells called astrocytes that support nerve cells that can occur in the brain or spinal cord. It can form in the brain or spinal cord. Despite the variety of modern therapies against GBM, it is still a deadly disease. Patients usually have a median survival of approximately 14 to 15 months from the diagnosis. Glioblastoma is also known as glioblastoma multiforme. The pathogenesis contributing to the proliferation and metastasis of cancer involves aberrations of multiple signalling pathways through multiple genetic mutations and altered gene expression. The coagulant factors like thrombin and tissue factor play a noteworthy role in cancer invasion. They are produced in the microenvironment of glioma through activation of protease-activated receptors (PARs) which are activated by coagulation proteases. PARs are members of family G-protein-coupled receptors (GPCRs) that are activated by coagulation proteases. These components play a key role in tumour cell angiogenesis, migration, invasion, and interactions with host vascular cells. Further, the release of neurotransmitters is also found to regulate malignancy in gliomas. Exploration of the interplay between malignant neural circuitry with the normal conditions is also decisive in finding effective therapies for these apparently invasive tumours. The present review discusses the molecular classification of gliomas, activation of PARs by coagulation protease, and its role in metastasis of gliomas. Further, the differential involvement of neurotransmitters in the pathogenesis of gliomas has also been discussed. Targeting these molecules may present a potential therapeutic approach for the treatment of gliomas.

Association of TNF-α Gene Expression and Release in Response to Anti-Diabetic Drugs from Human Adipocytes in vitro.

INTRODUCTION: TNF-α, a proinflammatory cytokine secreted by activated immune cells, and overexpression of it in adipocytes, has an important role in insulin resistance progression and diabetes development. AIM AND OBJECTIVE: Subcutaneous adipocytes derived from mesenchymal stem cells were used for in vitro analysis to find the role of antidiabetic drugs on TNF-α in high glucose-fed adipocytes. METHODS: In vitro adipocytes were used along with variable concentration of anti-diabetic drugs. The level of TNF-α was measured by ELISA and the mRNA level was quantified using SYBR-Green real-time PCR. All data were statistically analyzed. RESULTS: The level of TNF-α and the mRNA expression were observed and analyzed with normal adipocytes. TNF-α level and expression of it showed agonistic behavior, ie no change at low concentration while enhances with the increase of glucose. The level was decreased significantly when the adipocytes were treated with metformin (p=0.015) and pioglitazone (p=0.020). A combination of drugs showed that the expression of TNF-α was almost the same as for metformin alone. However, insulin increases the TNF-α expression as for pioglitazone. DISCUSSION: Such a report on adipocytes may be helpful for clinical benefits to understand the additional mechanism of adipocytes on the release and expression of TNF-α. However, anti-diabetic drugs including insulin up-regulate the TNF-α gene expression in mild or severe glucose load.

Regulation of HDAC1 and HDAC2 during consolidation and extinction of fear memory.

Histone deacetylases (HDACs) regulate gene expression epigenetically through synchronized removal of acetyl groups from histones required towards memory consolidation. Moreover, dysregulated epigenetic machinery during fear or extinction learning may result in altered expression of some of these genes and result in Post Traumatic Stress Disorder (PTSD). In the present study, region-specific expression of Histone deacetylase 1 (HDAC1) and Histone deacetylase 2 (HDAC2) was correlated to the acetylation of histones H3 and H4 and the resultant conditioned response, in rats undergone fear and extinction learning. The neuronal activation, histone acetylation at H3/H4 and expression of HDAC1/HDAC2 in centrolateral amygdala (CeL) and centromedial amygdala (CeM) of central Amygdala (CeA) and prelimbic (PL) and infralimbic (IL) of Prefrontal cortex (PFC) were found to be associated in a differential manner following fear and extinction learning. Moreover in CeM, the main output of the fear circuitry, the level of HDAC1 was down-regulated following conditioning and up-regulated following extinction as opposed to which HDAC2 was down-regulated in CeM following conditioning but not following extinction. Furthermore, in CeL the HDAC1 was upregulated and HDAC2 was downregulated following conditioning and extinction. This has important implications in speculating of the role of HDACs in fear memory consolidation and its extinction.

NMR-Based Serum Metabolomics Revealed Distinctive Metabolic Patterns in Reactive Arthritis Compared with Rheumatoid Arthritis.

Reactive arthritis (ReA) is a member of seronegative spondyloarthropathy (SSA), which involves an acute/subacute onset of asymmetrical lower limb joint inflammation weeks after a genitourinary/gastrointestinal infection. The diagnosis is clinical because it is difficult to culture the microbes from synovial fluid. Arthritis patients with a similar clinical picture but lapsed history of an immediate preceding infection that do not fulfill the diagnostic criteria of other members of SSA, such as ankylosing spondylitis, psoriatic arthritis, and arthritis associated with inflammatory bowel disease, are labeled as peripheral undifferentiated spondyloarthropathy (uSpA). Both ReA and uSpA patients show a strong association with class I major histocompatibility complex allele, HLA-B27, and a clear association with an infectious trigger; however, the disease mechanism is far from clear. Because the clinical picture is largely dominated by rheumatoid-arthritis (RA)-like features including elevated levels of inflammatory markers (such as ESR, CRP, etc.), these overlapping symptoms often confound the clinical diagnosis and represent a clinical dilemma, making treatment choice more generalized. Therefore, there is a compelling need to identify biomarkers that can support the diagnosis of ReA/uSpA. In the present study, we performed NMR-based serum metabolomics analysis and demonstrated that ReA/uSpA patients are clearly distinguishable from controls and further that these patients can also be distinguished from the RA patients based on the metabolic profiles, with high sensitivity and specificity. The discriminatory metabolites were further subjected to area under receiver operating characteristic curve analysis, which led to the identification of four metabolic entities (i.e., valine, leucine, arginine/lysine, and phenylalanine) that could differentiate ReA/uSpA from RA.

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy.

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)-obtained from joints affected in ReA-holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of 1 H and 13 C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) 1 H and 1 H─13 C NMR spectroscopy methods such as one-dimensional 1 H CPMG and two-dimensional J-resolved1 H NMR and homonuclear 1 H─1 H TOCSY and heteronuclear1 H─13 C HSQC correlation spectra. Compared with normal SF samples, several distinctive 1 H NMR signals were identified and assigned to metabolites in the 1 H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.

Decreased level of histone acetylation in the infralimbic prefrontal cortex following immediate extinction may result in deficit of extinction memory.

In the last few decades, there has been exponential increase in studies aiming to trace the molecular mechanism of fear extinction with a hope to minimize the return of fear after exposure therapy required for operational treatment of anxiety disorders. The present study explored how the timing of extinction training after developing a specific fear, affects the consequent return of the extinguished fear and the role of histone acetylation in controlling the circuitry, thereof. It was found that rats undergone extinction training 10 min. after fear memory acquisition (Immediate Extinction) had deficits in retention of extinction memory as compared to one which underwent extinction 24 h after fear acquisition (Delayed Extinction). When the differences were sorted at the circuitry level the relative activity of the infralimbic prefrontal cortex (IL) to prelimbic cortex (PL) was found to be lower in the immediate extinction group as compared to the delayed extinction group as evidenced by the c-fos expression in the mPFC of these groups. Further investigation showed that acetylation of histone H3/H4 along with the levels of CREB binding protein (CBP) which is a histone acetyltransferase (HAT), was associated with neuronal activation and was significantly lower in the IL of the immediate extinction group than the delayed extinction group. In conclusion, the observed deficits in the immediate extinction group may be the result of compromised activation of IL, which in turn may be associated with changes in histone acetylation.

Identification and characterization of Fusarium mangiferae as pathogen of mango malformation in India

ABSTRACT Fusarium mangiferae (=F. subglutinans) isolates collect from malformed samples from major mango-growing area of North India. Molecular identification and characterization of eleven most virulent isolates of F. mangiferae, based on pathogenicity tests used for the present study. Species-specific, genus specific ITS-PCR and PCR-RFLP performed for the accurate and easy detection of F. mangiferae. The rDNA-ITS 28S region sequences used for phylogenetic analysis of Fusarium isolates from India and other countries for homology search between them. The phylogenetic tree divided the isolates into three clades (i.e., American, Asian and African) and showed the high level of sequence based similarity (69-99%) among all Fusarium sequences from Asia. Thus, claimed Fusarium mangiferae as dominant pathogen of mango malformation. Furthermore, we conclude that exploiting the nested PCR coupled with PCR-RFLP will help in rapid and accurate detection of F. mangiferae pathogen of mango malformation.

Association of NAT2 gene polymorphisms with susceptibility to esophageal and gastric cancers in the Kashmir Valley.

BACKGROUND AND AIMS: The high incidence of gastrointestinal cancers in the Kashmir Valley has been attributed to the presence of many chemical carcinogens such as nitrosamines and heterocyclic amines in tobacco and salted tea. Due to functional polymorphisms of the N-acetyltransferase 2 (NAT2) gene, there may be interindividual differences in the metabolism of heterocyclic amines. We undertook this study to determine the influence of NAT2 gene polymorphisms (rs1799929, rs1799930, rs1799931) as well as their interactions with environmental carcinogens on the modulation of risk of esophageal and gastric cancers (EC and GC) in the Kashmir Valley. METHODS: A case/control study was performed involving 398 study subjects (182 controls, 123 EC and 93 GC). DNA samples were genotyped by PCR-RFLP method. RESULTS: None of the three NAT2 polymorphic alleles was found to be independently associated with risk of EC/GC but haplotypes C(481)A(590)G(857) and T(481)A(590)G(857) significantly modulated the risk of EC and GC, respectively (OR=0.56; 95% CI=0.34-0.91; p=0.018 and OR=4.61; 95% CI=1.90-11.17; p=0.001). NAT2 slow acetylator genotypes (NAT2 *5, NAT2 *6, NAT2 *7) significantly increased the risk of esophageal squamous cell carcinoma (ESCC, OR=1.73; 95% CI=1.01-2.97; p=0.047). Smoking and salted tea consumption were independent risk factors, but they did not show any interaction with NAT2 slow acetylator genotypes. CONCLUSIONS: NAT2 slow acetylator genotype may increase susceptibility to ESCC, and NAT2 haplotypes (C(481)A(590)G(857) and T(481)A(590)G(857)) may predict susceptibility to EC and GC in the Kashmir Valley.

MTHFR and ACE gene polymorphisms and risk of vascular and degenerative dementias in the elderly.

Focal lacunar infarctions due to cerebral small vessel atherosclerosis or single/multiple large cortical infarcts lead to vascular dementia, and different genes and environmental factors have been implicated in causation or aggravation of the disease. Previous reports suggest that some of the risk factors may be common to both vascular as well as degenerative dementia. Among genetic factors, role of angiotensin converting enzyme (ACE) and methylene-tetrahydrofolate reductase (MTHFR) genes as putative risk factors has been examined but the outcome of these studies remain inconclusive. Present study attempted to see the importance of ACE alu insertion/deletion and MTHFR C677T polymorphisms as genetic predisposers to dementia. The study comprised of 80 vascular dementia patients, 90 degenerative dementia patients and 170 age matched controls. All were genotyped for ACE, MTHFR and APOE polymorphisms using PCR-RFLP method. Frequency of ACE D allele was seemingly high in dementia cases (26.7%) when compared to controls (11.2%). However, after adjusting for age and APOE E4*, none of the ACE alleles showed good correlation. MTHFR genotypes or alleles also did not show any correlation. Our study suggests no true correlation of ACE or MTHR genes with dementia in elderly.

Genetic susceptibility of epidermal growth factor +61A>G and transforming growth factor beta1 -509C>T gene polymorphisms with gallbladder cancer.

Epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1) play important roles in tumor biology. Single nucleotide polymorphisms in EGF and TGFB1 genes alter the expression of these growth factors and influence the tumorigenesis process. The aim of our present study was to determine the association of EGF+61A>G (rs4444903) and TGFB1-509C>T (rs1800469) gene polymorphism with susceptibility to gallbladder cancer (GBC). The present case-control association study was carried out in 126 confirmed GBC patients and 190 healthy subjects. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism methods. The GG genotype of EGF+61A>G was significantly associated with GBC [p=0.012, odds ratio (OR)=2.22, 95% confidence interval (CI)=1.19-4.15] in comparison to healthy subjects. Analysis based on gender indicated risk due to GG genotype was limited to female GBC patients (p=0.003, OR=3.45, 95% CI=1.52-7.82). Upon stratification of GBC patients on the basis of the presence or absence of gallstones, the risk due to EGF polymorphism was not modulated by the status of gallstones. The TGFB1-509C>T polymorphism was not associated with GBC. Also, we did not find any association of this polymorphism when GBC patients were subdivided on the basis of gender. However, after stratification of GBC patients on the status of gallstones, we determined that the CT genotype of TGFB1 was associated with increased risk of GBC without gallstones (p value=0.030, OR=2.90, 95% CI=1.26-6.69). Furthermore, the combination of the GG genotype of EGF and the CT genotype of TGFB1 demonstrated synergistic increase in risk of GBC. In conclusion, the higher producing +61G allele of EGF and -509 CT genotype of TGFB1 synergistically increase the susceptibility of gallbladder cancer (p value=0.003). Further study in large samples size is required to confirm our findings.

The involvement of secondary signaling molecules in cytochrome P-450 1A1-mediated inducible nitric oxide synthase expression in benzo(a)pyrene-treated rat polymorphonuclear leukocytes.

Benzo(a)pyrene induces cytochrome P-450 1A1 (CYP1A1) expression in rat polymorphonuclear leukocytes (PMNs) that upregulates expression of inducible nitric oxide synthase (iNOS). In the present study, the involvement of secondary signaling molecules in CYP1A1-mediated augmentation of iNOS expression in benzo(a)pyrene-treated rat PMNs was investigated. PMNs were isolated from the peripheral blood of controls and benzo(a)pyrene-treated rats. The expression and/or activity of CYP1A1, iNOS, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and intracellular calcium ([Ca(2+)]i) concentrations were measured in control and benzo(a)pyrene-treated rat PMNs with and without alpha-naphthoflavone, aminoguanidine, genistein, pyrrolidine dithiocarbamate (PDTC), felodipine, or SB202190 pre-treatment. A significant elevation in CYP1A1 and [Ca(2+)]i was observed in benzo(a)pyrene-treated rat PMNs, which was significantly restored by alpha-naphthoflavone or genistein. Neither PDTC, SB202190, nor aminoguanidine altered the benzo(a)pyrene-mediated increase in [Ca(2+)]i. Although felodipine reduced the benzo(a)pyrene-mediated increase in [Ca(2+)]i, no significant change was observed in CYP1A1 expression and activity. Benzo(a)pyrene-augmented iNOS expression and activity in PMNs were significantly reverse by felodipine, genistein, or PDTC. Benzo(a)pyrene also induced TNF-alpha and IL-1beta production in PMNs, which was significantly reversed by genistein. The results demonstrated the involvement of [Ca(2+)]i, tyrosine kinase, inflammatory cytokines, and NF-kappaB in CYP1A1-mediated iNOS expression in benzo(a)pyrene-treated rat PMNs.

Slow acetylator genotype of N-acetyl transferase2 (NAT2) is associated with increased susceptibility to gallbladder cancer: the cancer risk not modulated by gallstone disease.

This study comprised 124 consecutive cases of proven GBC and 147 healthy controls. NAT2 polymorphisms were carried out using PCR-RFLP method. The NAT2 slow acetylator genotype was significantly associated with risk of GBC (OR 3.4, 95% CI =1.9-5.7 p = 0.000007). The NAT2 2*6 and 2*7 allele frequencies were higher in GBC and conferred significant risk of cancer (OR 1.9, 95% Cl = 1.2-2.9, p= 0.006; OR, 2.9, 95% Cl = 1.6-5.2, p = 0.0001). The haplotypes 2, 1, 1 and 1, 2, 1 were significantly higher (OR 3.7 95% Cl 2.1-7.0, p = 0.00001; OR 1.8 95% Cl =1.1-2.8, p = 0.008) in GBC. The risk of slow acetylator phenotype in GBC patients with or without gallstones was similar. These results suggest that NAT2 slow acetylator phenotype influences the susceptibility of gallbladder cancer and the risk is not modulated by gallstone disease.

Do TNFA -308 G/A and IL6 -174 G/C gene polymorphisms modulate risk of gallbladder cancer in the north Indian population?

OBJECTIVES: Gallbladder carcinoma (GBC) is highly aggressive neoplasm which arises in the background of gall stones and inflammation. GBC affects women 2-3 times more frequently than men. Pro-inflammatory TNFA and IL6 gene polymorphism has been associated with various inflammatory diseases. The aim of this study was to investigate whether TNFA -308 (G/A) and IL6 -174 G/C polymorphisms within flanking region of the genes are associated with GBC susceptibility. METHODS: The promoter polymorphisms were genotyped using PCR-RFLP in 200 healthy subjects and 124 GBC patients. RESULTS: Frequency distribution of TNFA -308 (G/A) and IL6 -174 G/C were not significantly different in GBC patients in comparison to healthy controls. However, frequency of TNFA -308 (G/A) polymorphism in female GBC patients without gallstone were significantly different (p-value= 0.006) when compared to healthy female subjects (OR=3.054; 95% CI=1.39-6.72). CONCLUSION: These results suggest that TNFA -308 (G/A) polymorphism may influence the susceptibility of female gender gallbladder cancer in absence of gallstones while IL6 -174 G/C polymorphism does not seem to be playing significant role in the susceptibility to gallbladder cancer.