X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.     

Cell proliferation and interferon-gamma response to recombinant MBP-3, NarL, MT-10.3, and 16 kDa Mycobacterium tuberculosis antigens in Brazilian tuberculosis patients

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-gamma (IFN-gamma)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-gamma human T cell immune responses, to four recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%) and controls (> 64%) respond to the PPD, 16 kDa was more specifically recognized (> 21%) although less sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average level of IFN-gamma was induced by 16 kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8 pg/ml x 221.6 pg/ml, respectively), although the difference was not significant. These data show that in contrast with the other recombinant proteins, the stimulatory potency of 16 kDa to induce proliferative and INF-gamma response was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a more selective immune response than PPD.     

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).     

Phylogenetic relationships among the baleen whales based on maternally and paternally inherited characters

Phylogenetic relationships in the Cetacean suborder Mysticeti (baleen whales) have recently been the focus of increased attention. Here, we examine the evolutionary history of this group by comparing genealogies derived from Y chromosome and mitochondrial DNA sequences. We generated topologies based on paternally and maternally inherited characters for males from nine baleen whale species, including representatives of three families (Balaenidae, Eschrichtiidae, and Balaenopteridae) and four genera (Balaena, Eschrichtius, Balaenoptera, and Megaptera). Divergence among species was fifteen times greater for mtDNA than for Y-specific DNA. Both mtDNA and yDNA topologies revealed the family Balaenopteridae to be paraphyletic, but this relationship was neither strongly supported nor consistent across phylogenetic analysis methodologies. Humpback and fin whales, representing different genera, were reciprocally monophyletic sister species according to mtDNA. Although the monophyly of fin whales decayed for yDNA, a close relationship between fin and humpback whales was retained in yDNA trees. The paraphyly of fin whales and the long branch leading to humpback whales for the yDNA marker may suggest life history differences between these species. Specifically, male humpback whales showed higher than average divergence from other baleen whales at yDNA, although not at mtDNA, suggesting a potential for smaller effective population sizes among male humpbacks on an evolutionary timescale. The observation that those species that have been found to hybridize in nature (blue/fin and blue/humpback) do not reveal evidence for paraphyly for either maternal or paternal markers suggests that introgressive hybridization has not historically been extensive and thus may not represent a substantial source of phylogenetic error for Mysticeti.     

Human blood plasma advanced oxidation protein products (AOPP) correlates with fibrinogen levels

In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP) was detected in the plasma of chronic uremic patients. The aim of the present studies was to find out that which plasma fraction(s) is responsible for AOPP reactivity. Thermal treatment of pooled samples of human citrate-plasma or EDTA-plasma at 50 degrees C resulted in a rapid and parallel loss of fibrinogen concentration and AOPP reactivity. On the basis of time course and t1/2 values following thermal treatment, AOPP was indistinguishable from fibrinogen. There was a statistically significant (p < 0.0001) correlation between levels of blood plasma fibrinogen and AOPP in patients (n = 61) with various peripheral vascular or cardiovascular diseases. There was also a significant (p < 0.0001) relationship between plasma levels of fibrinogen and molar AOPP/fibrinogen ratio indicating that higher fibrinogen concentrations were associated with more oxidatively transformed groups on the molecule. Results of the present studies suggest that post-translationally modified fibrinogen is a key molecule responsible for human plasma AOPP reactivity. It remains to be elucidated what is the pathophysiological significance of the post-translationally modified fibrinogen in the inflammation-associated events of atherosclerosis, in platelet aggregation, and as a cardiovascular risk biomarker.     

Transcriptomic analysis of the effect of ifosfamide on MDCK cells cultivated in microfluidic biochips

We investigated the behavior of renal cells cultivated in microfluidic biochips when exposed to 50 μM of ifosfamide, an antineoplastic drug treatment. The microarray analysis revealed that ifosfamide had any effect in Petri conditions. The microfluidic biochips induced an early inflammatory response in the MDCK in the untreated cells. This was attributed to cells adapting to the dynamics and micro environment created by the biochips. This led to modulations in the mitochondria dysfunction pathway, the Nrf-2 and oxidative stress pathways and some related cancer genes. When exposed to 50 μM of ifosfamide, we detected a modulation of the pathways related to the cancer and inflammation in the MDCK cultivated in the biochips via modulation of the ATM, p53, MAP Kinase, Nrf-2 and NFKB signaling. In addition, the genes identified and related proteins affected by the ifosfamide treatment in the biochips such as TXNRD1, HSP40 (DNAJB4 and DNAJB9), HSP70 (HSPA9), p21 (CDKN1A), TP53, IKBalpha (NFKBIA) are reported to be the molecular targets in cancer therapy. We also found that the integrin pathway was perturbed with the ifosfamide treatment. Finally, the MYC proto-oncogene appeared to be a potential bridge between the integrin signaling and the anti-inflammatory response.     

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.