SHP-1 Phosphatase Is a Critical Regulator in Preventing Natural Killer Cell Self-Killing

Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these "de-regulated" SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.     

Plant regeneration in <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> from cell suspension cultures

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1–3 × 10⁴ cells per cm³ of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm⁻³ benzyladenine (BA) along with 0.05 mg dm⁻³ α-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm⁻³ BA. These microshoots after dipping in 1–2 cm³ of 10 mg dm⁻³ indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80–82 % rooting within 4 weeks.     

Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury

In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.     

Exploring the DNA mimicry of the Ocr protein of phage T7

DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.     

ROS- and HIF1α-dependent IGFBP3 upregulation blocks IGF1 survival signaling and thereby mediates high-glucose-induced cardiomyocyte apoptosis

The prevalence of chronic hyperglycemia and its complications, imposing a critical burden on the worldwide economy and the global healthcare system, is a pressing issue. Mounting evidence indicates that oxidative stress and hypoxia, two noticeable features of hyperglycemia, play a joint crucial role in mediating cellular apoptosis. However, the underlying detailed molecular mechanism remains elusive. Triggered by the observation that insulin-like growth factor (IGF1)-binding protein 3 (IGFBP3) can mediate, in renal cells, high-glucose-induced apoptosis by elevating oxidative stress, we wish to, in this study, know whether or not the similar scenario holds in cardiac cells and, if so, to find its relevant molecular key players, thereby dissecting the underlying molecular pathway. Specifically, we used a combination of three different cellular sources (H9c2 cells, diabetic rats, and neonatal rat ventricular cardiomyocytes) as our model systems of study. We made use of Co-IP assay and western blot analysis in conjunction with loss-of-function reasoning, gain-of-function logic, and inhibitor treatment as our main analytical tools. As a result, briefly, our main findings are that hyperglycemia can induce cardiac IGFBP3 overexpression and secretion, that high levels of IGFBP3 can sequester IGF1 from IGF1 survival pathway, leading to apoptosis, and that IGFBP3 gene upregulation is hypoxia-inducible factor (HIF)1α-dependent and reactive oxygen species dependent. Piecing these findings together allows us to propose the improved molecular regulatory mechanism. In conclusion, we have established the molecular roles of IGFBP3, HIF1, and prolyl hydroxylase domain in connecting oxidative stress with hypoxia and in cellular apoptosis under hyperglycemia.     

Gallic acid‐based alkyl esters synthesis in a water‐free system by celite‐bound lipase of Bacillus licheniformis SCD11501

Gallic acid (3, 4, 5‐ trihydroxybenzoic acid) is an important antioxidant, anti‐inflammatory, and radical scavenging agent. In the present study, a purified thermo‐tolerant extra‐cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross‐linking agent, glutaraldehyde. The celite‐bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite‐bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n‐propyl gallate (72.1%), and n‐butyl gallate (63.8%) at 55^oC in 10 h under shaking (150 g) in a water‐free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and ¹H NMR spectrum analysis. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:715–723, 2015     

Isocitrate dehydrogenase of Bradyrhizobium japonicum is not required for symbiotic nitrogen fixation with soybean

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.     

Effects of Ceftiofur and Chlortetracycline Treatment Strategies on Antimicrobial Susceptibility and on tet(A), tet(B), and bla CMY-2 Resistance Genes among E. coli Isolated from the Feces of Feedlot Cattle

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla _CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (n = 289, median = 6; 95% CI = 4–8) compared with the tet(B) gene (n = 208, median = 3; 95% CI = 3–4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.     

Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure

The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe αG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.