Caffeine-induced locomotor activity: Possible involvement of GABAergic-dopaminergic-adenosinergic interaction

Caffeine (10–40 mg/kg, p.o.) enhanced locomotor activity (LA). Administration of GABA antagonist, bicuculline (0.5–1.0 mg/kg, i.p.), potentiated this caffeine-induced increase of LA, as well as LA of control rats. Treatment with the GABA agonist, muscimol (0.25–1 mg/kg, i.p.) or dopaminergic antagonist, haloperidol (0.25–1 mg/kg, i.p.) or muscarinic receptor blocker, atropine (3.75–5 mg/kg, i.p.), or inhibitor of acetylcholine esterase physostigmine (0.05–0.30 mg/kg, i.p.) or nicotine (0.5–1.5 mg/kg, i.p.) an nicotinic receptor agonist all decreased the LA of both caffeinetreated and control rats. Haloperidol-induced reduction in caffeine-induced increase in LA was found to be withdrawn with higher dose of caffeine. The dopamine agonist L-Dopa (75–150 mg/kg, p.o.) along with carbidopa (10 mg/kg, p.o.) increased the LA in control rats and potentiated the LA of caffeine treated rats. The haloperidol attenuated the bicuculline-induced increase in LA and atropine or physostigmine attenuated the bicuculline or L-Dopa+carbidopa-induced increase in LA in both caffeine treated and control rats when those drugs were administered concomitantly with bicuculline or L-Dopa+carbidopa. These results suggest that (a) the GABAergic system has direct role in the regulation of LA, and (b) caffeine potentiates LA by antagonism of the adenosine receptor and activation of the dopaminergic system which, in turn, reduces GABAergic activity through the reduction of cholinergic system.     

Mucosubstance histochemistry of Brunner's glands,pyloric glands and duodenal goblet cells in the ferret

Summary The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods.The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man.The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomycins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of db/db mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.     

Evolutionary insights from suffix array-based genome sequence analysis

Gene and protein sequence analyses, central components of studies in modern biology are easily amenable to string matching and pattern recognition algorithms. The growing need of analysing whole genome sequences more efficiently and thoroughly, has led to the emergence of new computational methods. Suffix trees and suffix arrays are data structures, well known in many other areas and are highly suited for sequence analysis too. Here we report an improvement to the design of construction of suffix arrays. Enhancement in versatility and scalability, enabled by this approach, is demonstrated through the use of real-life examples. The scalability of the algorithm to whole genomes renders it suitable to address many biologically interesting problems. One example is the evolutionary insight gained by analysing unigrams, bi-grams and higher n-grams, indicating that the genetic code has a direct influence on the overall composition of the genome. Further, different proteomes have been analysed for the coverage of the possible peptide space, which indicate that as much as a quarter of the total space at the tetra-peptide level is left un-sampled in prokaryotic organisms, although almost all tri-peptides can be seen in one protein or another in a proteome. Besides, distinct patterns begin to emerge for the counts of particular tetra and higher peptides, indicative of a 'meaning' for tetra and higher n-grams. The toolkit has also been used to demonstrate the usefulness of identifying repeats in whole proteomes efficiently. As an example, 16 members of one COG,coded by the genome of Mycobacterium tuberculosis H37Rv have been found to contain a repeating sequence of 300 amino acids.     

Homology model,molecular dynamics simulation and novel pyrazole analogs design of Candida albicans CYP450 lanosterol 14 α-demethylase,a target enzyme for antifungal therapy

Candida albicans infections and their resistance to clinically approved azole drugs are major concerns for human. The azole antifungal drugs inhibit the ergosterol synthesis by targeting lanosterol 14α-demethylase of cytochrome P450 family. The lack of high-resolution structural information of fungal pathogens has been a barrier for the design of modified azole drugs. Thus, a preliminary theoretical molecular dynamic study is carried out to develop and validate a simple homologous model using crystallographic structure of the lanosterol 14α-demethylase of Mycobacterium tuberculosis (PDB ID-1EA1) in which the active site residues are substituted with that of C. albicans (taxid 5476). Further, novel designed pyrazole analogs (SGS1-16) docked on chimeric 1EA1 and revealed that SGS-16 show good binding affinity through non-bonding interaction with the heme, which is different from the leading azole antifungals. The ADME-T results showed these analogs can be further explored in design of more safe and effective antifungal agents.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

MAP2 prevents protein aggregation and facilitates reactivation of unfolded enzymes.

It is well established that in addition to its functional role in cell motility, cell division and intracellular transport, cytoskeletal protein tubulin also possesses significant chaperone-like activity. In vitro studies from our laboratory showed that dimeric tubulin can prevent stress induced aggregation of substrate proteins, can resist thermal deactivation of enzymes and can also refold enzymes from their fully denatured state [Manna, T., Sarkar, T., Poddar, A., Roychowdhury, M., Das, K.P. & Bhattacharyya, B. (2001) J. Biol. Chem.276, 39742-39747]. Negative charges of the C-termini of both subunits of tubulin are essential for this chaperone-like property as the deletion of only beta-C-terminus or the binding of a 14-residue basic peptide P2 to the alpha-C-terminus completely abolishes this property [Sarkar, T., Manna, T., Bhattacharyya, S., Mahapatra, P., Poddar, A., Roy, S., Pena, J., Solana, R., Tarazona, R. & Bhattacharyya, B. (2001) Proteins Struct. Funct. Genet.44, 262-269]. Based on these results, one would expect that the microtubular proteins (MTP, tubulin with microtubular-associated proteins, i.e. MAPs bound to the C-terminus) should not possess any chaperone-like activity. To our surprise we noticed excellent chaperone-like activity of MTP. MTP prevents chemical and thermal aggregation of other proteins and can enhance the extent of refolding of fully unfolded substrate enzymes. Because MTP contains tubulin as well as several MAPs bound to the C-termini of tubulin, we fractionated and purified microtubular associated protein 2 (MAP2) and tau using phosphocellulose chromatography. Experiments with purified proteins demonstrated that it is the MAP2 of MTP that exhibits significant chaperone-like activity. This has been shown by the prevention of dithiothreitol-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase and regain of enzymatic activity during refolding of unfolded substrates. Tau, which shares a homologous C-terminal domain with MAP2, possesses no such activity.