Measurement of serum glycosylated proteins and glycosylated low density lipoprotein fraction in diabetes mellitus

A simple method was developed for estimating serum glycosylated protein levels using gel filtration with Bio-Gel P6 by determining the protein and sugar content in the void volume fraction. The glycosylated protein levels (GSP) correlated well with fasting blood sugar levels and glycosylated albumin level (G-ALB) determined by affinity chromatography with Blue Sepharose CL6B. The glycosylation level of heparin-citrate precipitable fraction of serum which predominantly contained low density lipoprotein (G-LDL) also correlated well with GSP and LDL-cholesterol levels. Significantly different values were obtained for GSP, G-ALB, and G-LDL between normals and diabetics.     

The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[³H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [³H]Adenosine derived from 5′-[³H]AMP hydrolysis was further transformed into [³H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [³H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Design of a helix-bundle cross-link: NMR and UV-visible spectroscopic analyses and molecular modeling of ring-oxidized retinals

In order to generate potential chemical cross-links for studying the chromophore binding site of bacteriorhodopsin and related helix-bundle proteins, MnO2 was used to oxidize all-trans-retinal's ring moiety. The structures and solution conformations of three ring-oxidized retinal analogues have been determined by using UV-visible absorption and 1H and 13C NMR spectroscopies, primarily with regard to (i) the introduction of a functional group at the ring end of the chromophore, (ii) the retention of the all-trans geometry of the polyenal side chain, and (iii) the torsional angle of the ring-polyenal bond. Analyses of their UV-visible absorption spectral parameters (lambda max, epsilon max, and vibrational fine structure) and NMR spectral parameters (1H-1H coupling constants, 1H and 13C NMR chemical shifts, and 1H homonuclear Overhauser effects) indicated the 4-oxo and the 2,3-dehydro-4-oxo derivatives both possess the twisted 6-s-cis conformation adopted by most six-membered ring analogues of retinal in solution or crystal. However, the alpha-dioxocyclopentenyl analogue exists in solution predominantly (70-80%) as the planar 6-s-trans conformer, similar to violerythrine chromophore analogues. In order to identify the minor solution forms, molecular modeling and geometry optimizations using the semiempirical molecular orbital method AM1 defined two additional symmetry-related minima at +/- 30-40 degrees in its C6-C7 torsional energy profile. Because the chromophores of bacterio- and halorhodopsins and sensory rhodopsins are bound as the 6-s-trans conformer [Harbison, G.S., Smith, S.O., Pardoen, J.A., Courtin, J.M.L., Lugtenburg, J., Herzfeld, J., Mathies, R.A., & Griffin, R.G. (1985) Biochemistry 24, 6955-6962; Baselt, D.R., Fodor, S.P.A., van der Steen, R., Lugtenburg, J., Bogomolni, R.A., & Mathies, R.A. (1989) Biophys. J. 55, 193-196], we suggest that the cyclopentenyl analogue's alpha-diketo function may be favorably positioned within the binding pocket and sufficiently reactive toward nucleophilic attack to cross-link an arginine located in or near the ring end of the chromophore cavity: Arg134 according to the current model of bacteriorhodopsin's tertiary structure [Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., & Downing, K.H. (1990) J. Mol. Biol. 213, 899-929] or Arg82 as postulated from an alternate model constructed primarily to accommodate the external point charge contribution to bacteriorhodopsin's opsin shift.     

Conditions for balanced polymorphism in the presence of differential delay in developmental time

The effect of differential development on the genetic properties of a population is studied. A specific model applicable to a wide variety of insect populations is used and the conditions under which the population is polymorphic when differential delay in developmental time is present are investigated. It is found that polymorphism is present for a very wide range of selection. The effect of the delay is to introduce frequency dependent factors and thus to maintain polymorphism even in the absence of heterosis. The results are applied to experimental data on the moth species Gonodontis bidentata and on the Canberra strain of the housefly, Musca domestica.     

Effects of con A and other lectins on pure 5'nucleotidase isolated from lymphocyte plasma membranes.

Inhibition of purified or membrane-bound 5′nucleotidase by various lectins was studied in lymphocytes from pig mesenteric lymph nodes. Con A or $\text{Lens culinaris}$ lectin LcH inhibited (75 %) purified 5′nucleotidase by a non-competitive process without cooperativity. Inhibition by these lectins of 5′ nucleotidase activity in whole lymphocytes, plasma membranes (untreated or solubilized) and LcH-receptor fraction displayed high positive cooperativity, reached higher level (90 %) and was of mixed type. An interaction between lectin receptors and 5′nucleotidase accounted for these differences. Wheat germ agglutinin (WGA) and divalent Con A which are not mitogenic for T lymphocytes had no effect on 5′nucleotidase; pokeweed mitogen (PWM), mitogen of T and B cells, was not inhibitor. When membrane proteins were cross-linked by glutaraldehyde, Con A inhibition of whole lymphocyte 5′nucleotidase presented the same properties as the purified enzyme. Possible correlation between 5′nucleotidase inhibition and lymphocyte stimulation is discussed.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-A resolution

The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-A resolution to an overall completeness of 91% with an R(sym) of 0.071. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions: a = 85.0 A, b = 99.8 A, c = 103.4 A. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (R(free) = 0.287) for all the data to 2.7-A resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe(3+), 2CO(2-)(3) ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two alpha-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly.     

Synthesis and Characterization of a Fluorescent Analogue of Cyclic di-GMP

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.     

Molecular Markers Reveal Only Two Mud Crab Species of Genus Scylla (Brachyura: Portunidae) in Indian Coastal Waters

The taxonomic ambiguity of the Indian mud crab (genus Scylla de Hann 1833) is still a cause of concern as several papers have been published with misleading identification. This is the first attempt to resolve the taxonomic uncertainty of the mud crab commonly available in Indian coastal waters using molecular genetic markers (ITS-1 and sequencing of COI gene) combined with traditional morphometry. Additionally, we developed a PCR method by which Indian mud crab species can be identified rapidly and effectively. The results clearly indicate that the green morph of the Indian mud crab is Scylla serrata and the brown morph is S. olivacea. The S. serrata commonly mentioned in the literature from India is S. olivacea; the S. tranquebarica noted by many Indian researchers should belong to S. serrata. Caution should be taken when interpreting or implementing the biological, molecular, and aquaculture data in the literature.