Effect of cell concentration on the rheology of glucoamylase fermentation broth

During the process optimisation of glucoamylase production by Aspergillus awamori, cell morphology was controlled at such a state that spore aggregation was completely prevented. Samples from five fermentations on complex media using either glucose or starch as carbon source were characterised with a Bohlin CS rheometer. The experimental data were conveniently described in terms of the power law model.     

Conformational analysis of biologically active truncated linear analogs of endothelin-1 using NMR and molecular modeling.

Some linear truncated analogs of endothelin-1 display potent agonistic activity at the ET(B) receptor, especially when the side chain of Trp21 is N-formylated. Then, the three-dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two-dimensional double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a beta-turn-like folding of the C-terminal segment Asp18-Trp21 as a probable key-factor for activity.     

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.     

Parasite-induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster

Larval hemolymph tyrosinase activity in Drosophila melanogaster was detected with high performance liquid chromatography with electrochemical detection. The enzyme hydroxylated L-tyrosine, and oxidized the diphenol substrates L-dopa and dopamine. In larvae of a selected immune-reactive strain the rates of tyrosine hydroxylation, dopa oxidation, and dopamine oxidation were markedly increased during the early stages of melanotic encapsulation of the eggs of the parasitic wasp Leptopilina boulardi. Tyrosinase activity was not modified in parasitized larvae of a selected susceptible strain of D. melanogaster, in which hosts the parasitoids developed unmolested. During the same period of parasitization, the amount of free tyrosine in immune reactive larvae was approximately three times higher than in susceptible hosts. These data indicate that the tyrosinase system of the immune reactive strain is activated during parasitization, and this results in the synthesis of some precursors which ultimately produce a melanotic and sclerotic capsule around the eggs of the parasite. Based on known genetic information of the enzyme system in Drosophila, it appears that at least two genes may be involved in the activation process, one associated with the proenzyme for monophenol oxidase activity, and the second with the proenzyme for diphenol oxidase activity.     

Temperature,deltamethrin-resistance status and performance measures of Plutella xylostella: complex responses of insects to environmental variables

1. Worldwide, the excessive use of insecticides has resulted in field-evolved insecticide-resistant populations of diamondback moth (DBM), Plutella xylostella. A deltamethrin-resistant DBM population from the field was divided into two subpopulations in the laboratory. One population (S-strain) was maintained with no further exposure to insecticides, whereas the other population (R-strain) was maintained under a regime of intermittent selection with deltamethrin. 2. Individuals from both strains were reared at constant temperatures in the range 10–35 °C in the absence of deltamethrin, and the effects of rearing temperature on various traits were investigated. At the time of experimentation, the R-strain was 20-fold more resistant to deltamethrin than the S-strain. 3. Temperature differentially affected developmental time, adult life span, pupal weight, and fecundity of both strains. Although both strains laid eggs after being reared at 10 °C, few of these eggs were fertile. The R-strain developed significantly faster than the S-strain. The integrated performance of the S-strain and R-strain was greatest at 25 and 15 °C, respectively. 4. The present study provides important information on the complexities of the outcomes of the interactions between ectotherms and temperature. Specifically, temperature-trait relationships may not be unimodal, and ectotherm genotypes (in this case insecticide-resistance status) and abiotic stresses can interact with unpredictable outcomes. 5. Current models predicting DBM population dynamics and relative abundance in different locations do not consider different thermal biologies of different genotypes. The present study shows the dramatic effects of environment on many parameters used in these models and will help to enhance their accuracy, and thus their utility.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.