Substrate specificity of CTP:phosphocholine cytidylyltransferase.

The specificity of CTP:phosphocholine cytidylyltransferase from rat liver for phosphorylated bases has been investigated. The apparent Km for phosphocholine was 0.17 mM. As the number of methyl substituents on the phospho-base decreased, the apparent Km increased: 4.0 mM for phosphodimethylethanolamine, 6.9 for phosphomonomethylethanolamine and 68.4 for phosphoethanolamine. The Vmax for the reaction was similar for phosphocholine (12.6 mumol/min per mg protein), phosphomonomethylethanolamine (13.5 mumol/min per mg protein) and phosphoethanolamine (9.2 mumol/min per mg protein). When phosphodimethylethanolamine was the substrate, the Vmax was 3-fold higher (40.3 mumol/min per mg protein). Phosphoethanolamine, phosphomonomethylethanolamine and phosphodimethylethanolamine were competitive inhibitors of the cytidylyltransferase when phosphocholine was used as substrate with Ki values of 18.5 mM, 9.3 mM and 1.5 mM, respectively. The results show that the cytidylyltransferase is highly specific for phosphocholine.     

Growth,maturation and survival in a laboratory-reared Rhabdocoelid Turbellarian,Macrostomum orthostylum (BRAUN 1885)

Growth, maturation and survival of a free living turbellarian Macrostomum orthostylum (BRAUN), from a brackish water fish-farm, were studied in the laboratory under a constant temperature of 24 °C. The worms tolerated a wide range of salinity (1 to 30‰). Maximum growth (total length) of 1000 μm was attained in 56 days with a mean growth rate of 15.7 μm d^-1. Minimum maturation time (7 days) and highest longevity (112 days) were recorded in 9%. salinity. Survival period was considerably longer at lower salinities (1 to 10‰) and showed negative relationship with higher salinities (11 to 30‰).     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)     

Salinity induced changes in the reproductive physiology of wheat plants

The effect of salinity on reproductive physiology of wheat wasinvestigated. One set of wheat plants was subjected to increasingsalt levels up to a certain concentration, whereas another setwas given the same concentration in a single application. Theformer was called "gradual" and latter "shock" treatment. Theireffects on pollen viability, germination and activity of starchsynthetase were studied. Gradual treatment seemed to reducethe toxic effects of salts on the viability of pollen grainsand their germination. Seeds obtained from the two sets weregerminated in the same salinities in which their plants hadbeen growing, and the results were compared with those of seedsobtained from control plants growing in a non-saline medium.The seeds of plants from the gradual treatment were better suitedfor germination on a saline medium than those from the shocktreatment or the control group. Salt treatment also increasedthe activity of starch synthetase at the midmilky stage in developinggrains. This phenomenon was considered essential for synthesisof starch in a saline environment. The increase in Na⁺ and Cl^– and decrease in K⁺ contentsof wheat grains in both treatments suggest that the effect ofsalinity on the physiological phenomenon studied is due to changesin the ionic content of the plants. ¹ In partial fulfilment of a Ph.D. degree from the Universityof Karachi, Pakistan. ² Professor of Botany, Director of Research Projects, Head,Plant Physiology Section, University of Karachi, Pakistan. (Received July 11, 1977; )     

Coassemble dopamine and GHK tripeptide into fluorescent nanoparticles for pH sensing

Fluorescent nanostructures have been widely applied to biomedical researches and clinical diagnosis such as biolabeling/imaging/sensing and have even acted as therapy reagents. Peptide‐based fluorescent nanostructures attract recent interest from biomedical researchers. Inspired by the natural existence of GHK‐Cu complex with a growth factor‐like effect in human blood, here we have developed a novel approach for designing nanosensors through the co‐assembling of two kinds of biomolecules. By making best use of both π‐π stacking between carbon rings and the easy‐oxidation property of an important transmitter molecule, dopamine (DA), we successfully built up a supersensitive and robust fluorescent pH nanosensor by co‐assembling oxidized DA (DA_ox) with a tripeptide GHK. The GHK‐DA_ox nanostructures have a quantum yield of 20.82%, which might be the brightest one among all the current co‐assembling structures merely through unmodified biomolecules. We envision this approach could open a new avenue for not only hybrid nanostructure construction, but also may inspire the bioengineering of in vivo luminescent probes.     

An Antioxidant Extract of Tropical Lichen,Parmotrema reticulatum,Induces Cell Cycle Arrest and Apoptosis in Breast Carcinoma Cell Line MCF-7

This report highlights the phytochemical analysis, antioxidant potential and anticancer activity against breast carcinoma of 70% methanolic extract of lichen, Parmotrema reticulatum (PRME). Phytochemical analysis of PRME confirms the presence of various phytoconstituents like alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, anthraquinones, and ascorbic acid; among which alkaloids, phenols and flavonoids are found in abundant amount. High performance liquid chromatography (HPLC) analysis of PRME revealed the presence of catechin, purpurin, tannic acid and reserpine. Antioxidant activity was evaluated by nine separate methods. PRME showed excellent hydroxyl and hypochlorous radical scavenging as well as moderate DPPH, superoxide, singlet oxygen, nitric oxide and peroxynitrite scavenging activity. Cytotoxicity of PRME was tested against breast carcinoma (MCF-7), lung carcinoma (A549) and normal lung fibroblast (WI-38) using WST-1 method. PRME was found cytotoxic against MCF-7 cells with an IC₅₀ value 130.03±3.11 µg/ml while negligible cytotoxicity was observed on A549 and WI-38 cells. Further flow cytometric study showed that PRME halted the MCF-7 cells in S and G2/M phases and induces apoptosis in dose as well as time dependent manner. Cell cycle arrest was associated with downregulation of cyclin B1, Cdk-2 and Cdc25C as well as slight decrease in the expression of Cdk-1 and cyclin A1 with subsequent upregulation of p53 and p21. Moreover PRME induced Bax and inhibited Bcl-2 expression, which results in increasing Bax/Bcl-2 ratio and activation of caspase cascade. This ultimately leads to PARP degradation and induces apoptosis in MCF-7 cells. It can be hypothesised from the current study that the antioxidant and anticancer potential of the PRME may reside in the phytoconstitutents present in it and therefore, PRME may be used as a possible source of natural antioxidant that may be developed to an anticancer agent.     

Small Hydrophobic Protein of Human Metapneumovirus Does Not Affect Virus Replication and Host Gene Expression In Vitro

Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.