Expression and purification of Arg196 and Lys272 mutants of mevalonate kinase from Methanococcus jannaschii

In microorganisms and plants, mevalonate kinase is involved in the biosynthesis of isoprenoid derivatives, one of the largest groups of natural products. We subcloned the gene of mevalonate kinase from Methanococcus jannaschii into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene. A variety of mutant expression plasmids including pMMK(R196K), pMMK(R196Q), pMMK(R196V), pMMK(K272R), and pMMK(K272A) have been constructed using site-directed mutagenesis. The wild-type protein and mutants were overexpressed and purified with a nickel HiTrap chelating metal affinity column to homogeneity. CD spectroscopy of wild-type protein and mutants indicates that none of the above mutations induces significant secondary structural changes. The results from kinetic studies showed that Arg196 is an essential residue for the function of the enzyme. Kinetic studies of Lys272 mutants indicate that salt bridge Lys272-Glu14 plays an important role in maintaining the active site microenvironment that is essential for catalytic activity of the enzyme.     

Effect of modified diabetic splenocytes on mice injected with multiple low-dose streptozotocin

This work reports the effects of a previous injection of mitomycin-modified splenocytes from multiple-low dose streptozotocin-treated mice (mld-sz) on autoimmune diabetes produced by mld-sz. Our work shows that a previous inoculation of modified mononuclear splenocytes from mld-sz mice prevents alterations in glycemia, in insulin secretion (IS) pattern from isolated perifused islets, and in mass of pancreatic islets. Immunohistochemistry showed an alteration in the number of beta, but not of alpha or delta cells. While a mononuclear intra-islet infiltration was observed in mld-sz mice, a predominantly polar or peri-islet infiltration was seen in vaccinated mice. Islet-associated mononuclear cells from mld-sz mice produced diabetes and induced a diminished IS when transferred to normal receptors. Those cells from previously vaccinated mld-sz mice had no effect when injected into normal receptors. In addition, they also inhibited the damage induced in normal receptors by the islet-associated mononuclear cells from mld-sz animals. Cellular death was also prevented by previous vaccination. Our results suggest that vaccination with modified splenocytes from mld-sz mice is capable of shifting the islet cells infiltration pattern from an aggressive one toward a protective one and thus preventing the beta cell destruction observed in mld-sz mice.     

Phenotypic plasticity to drought in seedlings of the warm season grass Panicum coloratum is related to collection site

Phenotypic plasticity is the ability of organisms to modify their phenotype in response to environmental changes. We estimated and compared the amount of phenotypic plasticity in response to drought in seedlings of different accessions of two varieties (var. makarikariense and var. coloratum) of Panicum coloratum, an allogamous warm season perennial grass, introduced and collected in sites in Argentina with different precipitation regimes. Amount of phenotypic plasticity was quantified in shoot/root biomass, blade/sheath biomass, specific leaf area and leaf area ratio (leaf area/total biomass) and mean phenotypic plasticity was estimated. The two genetically distinct varieties differed in the phenotypic plasticity of leaf area ratio (p = 0.008, F‐test), with var. makarikariense showing higher phenotypic plasticity. Accessions within varieties differed in phenotypic plasticity of leaf area ratio, specific leaf area, blade/sheath biomass and mean phenotypic plasticity (p < 0.05, F‐test). A strong relationship (r = 0.82, p < 0.01, F‐test) between mean phenotypic plasticity of each accession and precipitation variability was found. Relationships between phenotypic plasticity of blade/sheath biomass and leaf area ratio with annual mean precipitation were r = 0.86 and r = 0.75, respectively (p < 0.05; p < 0.01, F‐test, respectively). Evidence of a decoupling between phenotypic plasticity of above‐ versus belowground characters was apparent; outcomes on the interpretation of the variability in phenotypic plasticity and the potential applications of this variability are presented.     

Nanoparticles that display short collagen-related peptides. Potent stimulation of human platelet aggregation by triple helical motifs

Collagen plays a key role in the activation and adhesion of blood platelets via their cell-surface receptors. Normally, collagen-related peptides (CRPs), even one as long as a 30-mer (10 Gly-Pro-Hyp (GPO) repeats), are unable to effectively express collagen's platelet-activating behavior. We attached two short CRPs, AcHN-(Gly-Pro-Hyp)nGly-OH with n = 5 (1) and n = 10 (2), via the C-terminus to amino-functionalized latex nanoparticles to create a multimeric display of triple helical motifs. These nanomaterials were characterized by dynamic light scattering and environmental scanning electron microscopy. The nanoparticles bearing the 31-mer CRP sequence, 2, but not the 16-mer sequence, 1, effectively induced the aggregation of human platelets, with a potency level approaching that of native type I collagen. Our results highlight the importance of presenting triple helical CRP motifs of sufficient length on a suitable scaffold in order to stimulate platelets.     

Hydrophobic vs coulombic interactions in the binding of steroidal polyamines to DNA

Seven new steroidal polyamines derived from bile acids, either lithocholic or deoxycholic acid, have been studied as DNA-binding agents using four complimentary methods: an ethidium displacement assay, observed changes in the thermal denaturation of poly[d(AT)], effects on hyperchromicity of DNA, and circular dichroism. In addition, modelling studies were conducted to examine the electrostatic surface potential of the polycations. The results point to a key role for a large hydrophobic surface area on the steroid in addition to the Coulombic attraction by ammonium and guanidinium groups on the steroid interacting with the polyphosphate backbone.     

Synthesis of CDP-diglyceride from phosphatidylinositol and CMP

A reaction in which CDP-diglyceride and inositol are formed from 1-stearoyl, 2-arachidonoyl phosphatidylinositol and CMP occurs readily in dialyzed microsomal preparations from the mouse pancreas. The reaction is Mn²⁺-dependent, and it is inhibited by each of the two products, CDP-diglyceride and myoinositol. It is presumed to involve the back-reaction of CDP-diglyceride: inositol phosphatidyltransferase (phosphatidyl-inositol synthetase, EC.2.7.8.11.)     

The vesicular integral protein-like gene is essential for development of a mechanosensory system in zebrafish

The zebrafish hi472 mutation is caused by a retroviral insertion into the vesicular integral protein-like gene, or zVIPL, a poorly studied lectin implicated in endoplasmic reticulum (ER)-Golgi trafficking. A mutation in the shorter isoform of zVIPL (zVIPL-s) results in a reduction of mechanosensitivity and consequent loss of escape behavior. Here we show that motoneurons and hindbrain reticulospinal neurons, which normally integrate mechanosensory inputs, failed to fire in response to tactile stimuli in hi472 larvae, suggesting a perturbation in sensory function. The hi472 mutant larvae in fact suffered from a severe loss of functional neuromasts of the lateral line mechanosensory system, a reduction of zVIPL labeling in support cells, and a reduction or even a complete loss of hair cells in neuromasts. The Delta-Notch signaling pathway is implicated in cellular differentiation of neuromasts, and we observed an increase in Notch expression in neuromasts of hi472 mutant larvae. Treatment of hi472 mutant larvae with DAPT, an inhibitor of Notch signaling, or overexpression of the Notch ligand deltaB in hi472 mutant blastocysts produced partial rescue of the morphological defects and of the startle response behavior. We conclude that zVIPL-s is a necessary component of Delta-Notch signaling during neuromast development in the lateral line mechanosensory system.     

Partial purification of the aminopeptidase from the midgut of the human body louse, Pediculus humanus humanus

The midgut of the human body louse Pediculus humanus humanus contains a thermally stable leucine aminopeptidase, which was detected by agarose gel electrophoresis using l ‐amino oxidase. Midgut extracts were homogenized in saline or in 1% Triton X‐100 and the aminopeptidase was purified by Superose 6 gel filtration chromatography. A peak with enzyme activity that was extracted with or without Triton X‐100 was eluted at a molecular weight 67–69 kDa. Non‐denaturing polyacrylamide gel electrophoresis resolved one band of molecular weight of 69 kDa for samples that were extracted in a saline buffer. Two closely linked bands of molecular weight 67 kDa and 69 kDa were observed in samples that were extracted in 1% Triton X‐100.