Molecular and Cellular Biochemistry

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, K m for the substrate (acetylthiocholine), was found to be 0.216 mM and K i for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.

A survey of the existing data on the interactions of glycolytic enzymes with the cellular structure in mammalian tissues has substantiated the occurrence of an extensive degree of such associations in all tissues and during all stages of development. Furthermore, a considerable specificity was evident between the individual multiple forms of the enzymes in relation to these associations. In reviewing these data, a model has been developed which proposes that the glycolytic sequence is best described as consisting of a number of segments in vivo, each segment formed by a cluster of isozymes, many of which can interact with the actin containing filaments of the cytomatrix. The novel features of this segmentation and compartmentation have been described, and evidence has been provided that these phenomena collectively play a key role in meeting the different types of energy requirement in the cytoplasm of divergent cell types, with the wide selection of isozymes in this system offering the potential for increased flexibility and control in this important area of metabolism.

Activation of epithelial-AR signaling is identified as the major cause of hyperproliferation of the cells during benign and malignant prostate conditions. However, the contribution of stromal-AR is also precarious due to its secretory actions that contribute to the progression of benign and malignant tumors. The present study was aimed to understand the influence of stromal-AR mediated actions on epithelial cells during BPH condition. The secretome (conditioned media-CM) was collected from AR agonist (testosterone-propionate-TP) and antagonist (Nilutamide-Nil) treated BPH patient-derived stromal cells and exposed to BPH epithelial cells. Epithelial cells exhibited increased cell proliferation with the treatment of CM derived from TP-treated stromal cells (TP-CM) but did not support the clonogenic growth of BPH epithelial cells. However, CM derived from Nil-treated stromal cells (Nil-CM) depicted delayed and aggressive BPH epithelial cell proliferation with increased clonogenicity of BPH epithelial cells. Further, decreased AR levels with increased cMyc transcripts and pAkt levels also validated the clonogenic transformation under the paracrine influence of inhibition of stromal-AR. Moreover, the CM of stromal-AR activation imparted positive regulation of basal/progenitor pool through LGR4, β-Catenin, and ΔNP63α expression. Hence, the present study highlighted the restricted disease progression and retains the basal/progenitor state of BPH epithelial cells through the activation of stromal-AR. On the contrary, AR-independent aggressive BPH epithelial cell growth due to paracrine action of loss stromal-AR directs us to reform AR pertaining treatment regimes for better clinical outcomes.

Owing to its poor prognosis, the World Health Organization (WHO) lists lung cancer on top of the list when it comes to growing mortality rates and incidence. Usually, there are two types of lung cancer, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), which also includes adenocarcinoma, squamous cell carcinoma and large cell carcinomas. ARF, also known in humans as p14ARF and in the mouse as p19ARF, is a nucleolar protein and a member of INK4, a family of cyclin-independent kinase inhibitors (CKI). These genes are clustered on chromosome number 9p21 within the locus of CDKN2A. NSCLC has reported the role of p14ARF as a potential target. p14ARF has a basic mechanism to inhibit mouse double minute 2 protein that exhibits inhibitory action on p53, a phosphoprotein tumour suppressor, thus playing a role in various tumour-related activities such as growth inhibition, DNA damage, autophagy, apoptosis, cell cycle arrest and others. Extensive cancer research is ongoing and updated reports regarding the role of ARF in lung cancer are available. This article summarizes the available lung cancer ARF data, its molecular mechanisms and its associated signalling pathways. Attempts have been made to show how p14ARF functions in different types of lung cancer providing a thought to look upon ARF as a new target for treating the debilitating condition of lung cancer.

Cyclooxygenase-2 (COX-2), one isoform of cyclooxygenase proinflammatary enzymes, is a causal factor for tumor development, invasion, metastasis, and chemoresistance. It is frequently overexpressed in a variety of human malignancies, including laryngeal carcinoma. To investigate its possibility as a therapeutic target for the treatment of laryngeal carcinoma, we employed RNA interference technology to downregulate endogenous gene COX-2 expression in laryngeal carcinoma cells and analyzed its phenotypical changes. Results showed that shRNA-mediated downregulation of COX-2 expression in human laryngeal carcinoma cells significantly inhibited cell proliferation and colony formation in vitro and reduced the potential of tumorigenicity in vivo. The specific downregulation led to cell arrest in the G0/G1 phase of cell cycle and final apoptosis induction. The increased apoptosis was associated with the ratios of Bcl-2 or Bcl-xL/Bax. In the present study, we also observed that the downregulation of COX-2 could obviously enhanced the cytotoxic effect of Taxanes both in vitro and in vivo. All these results suggest that knockdown of COX-2 expression can lead to potent antitumor activity and chemosensitizing activity to taxanes in human laryngeal carcinomas.

Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following32P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)16 simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.

The hyperthyroid state is associated with low hepatic glycogen levels, but paradoxically with a high activity of glycogen synthase and low activity of glycogen phosphorylase. We determined the effects of triiodo-L-thyronine (T3) on glycogen synthesis and glycogen synthase activity in rat hepatocytesin vitro. Culture of rat hepatocytes with T3 (100 nM–1 μM) for 16 h–40 h increases glycogen synthesis from glucose and gluconeogenic precursors. The stimulation of glycogen synthesis by T3 was associated with an increase in the activity of glycogen synthase and was additive with the long-term effects of insulin but not with the short-term stimulation of glycogen synthesis by insulin. Culture of hepatocytes with T3 (at concentrations up to 1 μM) did not affect the responsiveness of glycogen synthesis to short-term stimulation by insulin but culture with 10 μM-T3 decreased the responsiveness to insulin without affecting the basal rate. It is suggested that the high activity of glycogen synthase in the hyperthyroid state is due to a direct effect of T3 on the hepatocyte, but the low hepatic glycogen content is probably due to either secondary metabolite and/or endocrine changes or to impaired responsiveness to insulin. T3 may have an anabolic role in the control of hepatic glycogen storage in the euthyroid postprandial state. (Mol Cell Biol120: 151–158, 1993)