Apaf-1 overexpression partially overcomes apoptotic resistance in a cisplatin-selected HeLa cell line

Inhibition of caspase-3-mediated apoptosis has been hypothesized to be associated with chemoresistance. Investigations of apoptosis revealed that cytosolic cytochrome c is associated with a complex of apoptotic protease activating factor-1 (Apaf-1), an adapter molecule, and caspase-9 to activate caspase-3. However, whether these apoptotic molecules are involved in acquired cisplatin resistance is not understood. The present work shows reduced activation of caspase-3 and apoptosis in a cisplatin-selected HeLa cell line. Ac-DEVD-CHO, a caspase-3 inhibitor, inhibited cisplatin-induced apoptosis about 60-70% in both cell lines. Ac-LEHD-CHO, a caspase-9 inhibitor or Ac-IETD-CHO, a caspase-8 inhibitor, inhibited cisplatin-induced caspase-3 activation and apoptosis similarly in both cell lines. In addition, cisplatin induced the activation of caspase-9, the upstream activator of caspase-3, in a dose-dependent manner, and the activation of caspase-9 was less induced in resistant cells. The accumulation of cytosolic cytochrome c, an activator of caspase-9, and the induction of the mitochondrial membrane-associated voltage-dependent anion channel were also reduced in cisplatin-resistant cells. However, the concentration of Bcl-2 family proteins in cisplatin-resistant cells was normal. The concentration of Apaf-1 was unaltered in both cell lines. Increasing the cellular concentration of Apaf-1 through the transient expression of the gene increased the induction of apoptosis in resistant cells, associated with enhanced activation of caspase-9, caspase-3 and DNA fragmentation factor. Regression analysis reveals that the modification factor, the ratio of the slope in the linear range of the dose-response curve with Apaf-1 to the slope without Apaf-1, is 1.5 and 4.75 in the HeLa and cisplatin-resistant HeLa cells, respectively. These results indicate that apoptosis and caspases are less induced in cisplatin-selected HeLa cells. They also suggest that ectopic overexpression of Apaf-1 may partially reverse the acquired cisplatin resistance.     

The collaborative study on the genetics of alcoholism: Genetics

This review describes the genetic approaches and results from the family-based Collaborative Study on the Genetics of Alcoholism (COGA). COGA was designed during the linkage era to identify genes affecting the risk for alcohol use disorder (AUD) and related problems, and was among the first AUD-focused studies to subsequently adopt a genome-wide association (GWAS) approach. COGA's family-based structure, multimodal assessment with gold-standard clinical and neurophysiological data, and the availability of prospective longitudinal phenotyping continues to provide insights into the etiology of AUD and related disorders. These include investigations of genetic risk and trajectories of substance use and use disorders, phenome-wide association studies of loci of interest, and investigations of pleiotropy, social genomics, genetic nurture, and within-family comparisons. COGA is one of the few AUD genetics projects that includes a substantial number of participants of African ancestry. The sharing of data and biospecimens has been a cornerstone of the COGA project, and COGA is a key contributor to large-scale GWAS consortia. COGA's wealth of publicly available genetic and extensive phenotyping data continues to provide a unique and adaptable resource for our understanding of the genetic etiology of AUD and related traits.     

Optimization of sodium orthovanadate to treat streptozotocin-induced diabetic rats

Oral administration of sodium orthovanadate restored blood glucose to normal levels in streptozotocin-induced diabetic rats. To establish the safety dose and to evaluate the side effects of over dose, if any, different doses of vanadium were used in the present study. Low concentrations of vanadium (0.1 and 0.3 mg/ml in drinking water) restored blood glucose, urea, cholesterol and the status of liver pathophysiological enzymes to normal levels in experimental rats. High vanadate treatment proved to be toxic not only to diabetic but also to normal rats as evidenced from the observations on the blood urea, plasma and liver glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. Low vanadate treatment restored body homeostasis of diabetic rats and was found to be nontoxic to normals.     

Magic angle spinning NMR-based metabolic profiling of head and neck squamous cell carcinoma tissues

High-resolution magic-angle spinning (HR-MAS) proton NMR spectroscopy is used to explore the metabolic signatures of head and neck squamous cell carcinoma (HNSCC) which included matched normal adjacent tissue (NAT) and tumor originating from tongue, lip, larynx and oral cavity, and associated lymph-node metastatic (LN-Met) tissues. A total of 43 tissues (18 NAT, 18 Tumor and 7 LN-Met) from 22 HNSCC patients were analyzed. Principal Component Analysis of NMR data showed a clear classification between NAT and tumor tissues, however, LN-Met tissues were classified among tumor. A partial least-squares discriminant analysis model generated from NMR metabolic profiles was used to differentiate normal from tumor samples (Q(2) > 0.80, Receiver Operator Characteristic area under the curve >0.86, using 7-fold cross validation). HNSCC and LN-Met tissues showed elevated levels of lactate, amino acids including leucine, isoleucine, valine, alanine, glutamine, glutamate, aspartate, glycine, phenylalanine and tyrosine, choline containing compounds, creatine, taurine, glutathione, and decreased levels of triglycerides. These elevated metabolites were associated with highly active glycolysis, increased amino acids influx (anaplerosis) into the TCA cycle, altered energy metabolism, membrane choline phospholipid metabolism, and oxidative and osmotic defense mechanisms. Moreover, decreased levels of triglycerides may indicate lipolysis followed by β-oxidation of fatty acids that may exist to deliver bioenergy for rapid tumor cell proliferation and growth.     

Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+) and CD11b(+) cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(-) Sca-1(+)c-Kit(+) (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.     

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.     

The collaborative study on the genetics of alcoholism: Brain function

Alcohol use disorder (AUD) and related health conditions result from a complex interaction of genetic, neural and environmental factors, with differential impacts across the lifespan. From its inception, the Collaborative Study on the Genetics of Alcoholism (COGA) has focused on the importance of brain function as it relates to the risk and consequences of alcohol use and AUD, through the examination of noninvasively recorded brain electrical activity and neuropsychological tests. COGA's sophisticated neurophysiological and neuropsychological measures, together with rich longitudinal, multi-modal family data, have allowed us to disentangle brain-related risk and resilience factors from the consequences of prolonged and heavy alcohol use in the context of genomic and social-environmental influences over the lifespan. COGA has led the field in identifying genetic variation associated with brain functioning, which has advanced the understanding of how genomic risk affects AUD and related disorders. To date, the COGA study has amassed brain function data on over 9871 participants, 7837 with data at more than one time point, and with notable diversity in terms of age (from 7 to 97), gender (52% female), and self-reported race and ethnicity (28% Black, 9% Hispanic). These data are available to the research community through several mechanisms, including directly through the NIAAA, through dbGAP, and in collaboration with COGA investigators. In this review, we provide an overview of COGA's data collection methods and specific brain function measures assessed, and showcase the utility, significance, and contributions these data have made to our understanding of AUD and related disorders, highlighting COGA research findings.