Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Identification of amino compounds synthesized and translocated in symbiotic Parasponia

We investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non-legumes known to form a root nodule symbiosis with rhizohia. In the xylem sap of P. andersonii we identified asparagine. aspartate. glutamine, glutamated significant quantities of a non-protein amino acid. 4-methylglutamte(2-amino-4-methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (El) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4-methyleneglutamate and 4-methyleneglulamine. Using ¹⁵N₂ labelling and GC-Ms analysis of root nodule extracts we followed N₂ fixation and ammonia assimilation in P. andersonii root nodules and observed Label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of ¹⁵N into 4-methylglutamate in nodules, demonstrating the de nova synthesis of this non protein amino acid and suggesting a role in the translation of N in symbioticParasponia.     

Chromosomal and cell size analysis of cold tolerant maize

Summary C-band number, guard cell length, and chloroplast number per guard cell were determined for eight maize populations. These populations consisted of maize selected for cold tolerance at the University of Nebraska as well as the original unselected populations. The genome size of these populations had previously been determined. C-band number fluctuated concertedly with the changes in genome size indicating that deletions and additions of constitutive heterochromatin occurred during selection, resulting in altered genome sizes. Guard cell size of all the cold tolerant populations was greater than the cell size of the respective nonselected populations. Chloroplast number per guard cell was also higher in all the cold tolerant populations than in their parental populations, but the increases were not statistically significant. The results indicate that changes in genome size that occurred during selection for cold tolerance are the result of changes in amounts of C-band heterochromatin and that the selection process results in an increase in cell size in the cold tolerant populations.