Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.     

Inducible expression of amplified human beta interferon genes in CHO cells.

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.     

A variable region of the Sugarcane Bacilliform Virus (SCBV) genome can be used to generate promoters for transgene expression in sugarcane

Four promoters derived from sugarcane bacilliform virus (SCBV) were compared and characterised. Three were obtained by PCR amplification of purified virion DNA extracted from three sugarcane cultivars. The fourth promoter was obtained by subcloning from an almost genome-length clone of SCBV. All promoters were able to drive stable expression of -glucuronidase in sugarcane. The PCR-derived promoter sequences shared more DNA homology with banana streak virus than to the subcloned SCBV. The subcloned promoter was the strongest expressing and was able to drive reporter gene expression in vitro and in the leaves, meristems and roots of glasshouse-grown sugarcane. Expression levels were at least equal to or higher than those measured for the maize polyubiquitin promoter.     

Efficient expression of vip184DeltaP gene under the control of promoters plus Shine-Dalgarno (SD) sequences of cry genes from Bacillus thuringiensis

AIMS: To compare vip184DeltaP gene expression time course and Vip184 protein yield under the control of promoters and Shine-Dalgarno (SD) sequences of vip184, cry3A and cry1A gene from Bacillus thuringiensis respectively. METHODS AND RESULTS: Derived from the shuttle vector pHT3101, recombinant plasmids pHPT3, pHTP3A(Delta)P and pHTP1A(Delta)P were constructed with the native vip184 gene and the vip184(Delta)P gene, either under the control of promoters and SD sequences of cry3A or cry1A genes. When the above plasmids were transformed into an acrystalliferous B. thuringiensis strain Cry(-)B, their expression time course were consistent with those of vip184, cry3A and cry1A gene respectively. The maximum yields of Vip184 protein were increased when under the control of promoters plus SD sequences of cry3A and cry1A gene. CONCLUSIONS: The results showed that both cry3A and cry1A promoter/SD sequence combinations were able to enhance synthesis of Vip184 and change its expression time course. SIGNIFICANCE AND IMPACT OF THE STUDY: Both cry3A and cry1A promoter/SD systems offer a method for improving the expression efficacy of the vip184 gene in B. thuringiensis and it is possible to co-express the vip184 gene and cry genes and accumulate Vip184 in the form of inclusion bodies by these systems in order to construct novel useful B. thuringiensis engineered strains.     

Gamma interferon is able to enhance the oxidative metabolism of human neutrophils

The oxidative metabolism of human neutrophils has been studied after incubation of the cells with recombinant interferon-y. Neutrophils incubated for 2-4 hours with 2-50 U/ml recombinant interferon-y undergo a higher respiratory burst measured both as Oz consumption and Oz- production when stimulated with formyl-methionyl-leucyl-phenylalanine, Concanavalin A or phorbol myristate acetate. The potentiating effect of interferon-y requires more than one hour of incubation, is optimal at 20-50 U/ml and depends on the presence of serum in the incubation medium. The interferon effect depends on new protein synthesis. Cycloheximide at doses which do not alter the respiratory response of normal cells completely prevents the potentiating effect of interferon.     

Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa

In an attempt to identify valuable markers for potential freezeability of the equine spermatozoa, three ejaculates were collected from five Andalusian stallions and frozen using a standard protocol. Before freezing, three apoptotic cell markers were studied by flow cytometry (early changes in sperm membranes, mitochondrial membrane potential and caspase activity). Post-thaw, spermatozoa were again evaluated for these parameters. Sperm kinematics using CASA were also studied before and after freezing and thawing. Receiving operating system curves were used to evaluate the relative value of the apoptotic markers herein studied, as forecast for potential freezeability. From all parameters studied, the outcome of JC-1 (as proportion of spermatozoa showing simultaneously orange and green fluorescence) had the highest diagnostic power. For potentially bad freezers (less than 25% of intact spermatozoa post-thaw), the significant area under the ROC-curve was 0.985, with a 100% sensitivity and 99.8% specificity for a cut off value of 55.7.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells (K_d = 20.9 nM at 4 °C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.     

Identification, expression, and purification of a unique stable domain from human HSPC144 protein

HSPC144 is a newly identified gene in human CD34(+) hematopoietic stem/progenitor cells. In this work, we have expressed and purified the 225-residue protein from Escherichia coli BL21 (DE3) and identified a stable fragment HSPC144-P (residues 44-225) by limited proteolysis method. The HSPC144-P fragment exhibits high stability with a little increase of secondary structure percentage as compared with the full-length protein. We anticipated that the N-terminally truncated protein possesses a more compact structure. By sequence analysis, the proteolytic fragment shares a great similarity with DUF589 domain, a previously identified domain with unknown function. This novel domain is highly conserved in Thy28 proteins and is worthy of structural and functional studies. We have subcloned this homologous domain from HSPC144 protein and purified to homogeneity for structure analysis. The (15)N and (15)N/(13)C-labeled DUF589 domain samples have been prepared successfully and determination of the NMR structure is in progress.