Dissection of a human septin: definition and characterization of distinct domains within human SEPT4

The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.     

Molecular analysis of Arachis interspecific hybrids

Incorporation of genetic resistance against several biotic stresses that plague cultivated peanut, Arachis hypogaea (2n=4x=40), is an ideal option to develop disease resistant and ecologically safe peanut varieties. The primary gene pool of peanut contains many diploid wild species (2n=2x=20) of Arachis, which have high levels of disease and insect resistances. However, transfer of resistant genes from these species into A. hypogaea is difficult due to ploidy level differences and genomic incompatibilities. This study was conducted to monitor alien germplasm transmission, using Random Amplified Polymorphic DNA (RAPD) markers, from two diploid wild species, A. cardenasii and A. batizocoi, into A. hypogaea. Triploid interspecific hybrids were produced by crossing two A. hypogaea cultivars (NC 6 and Argentine) with the two species and by colchicine-treating vegetative meristems, fertility was restored at the hexaploid (C_o) level in the four hybrids. Hexaploids were allowed to self-pollinate for four generations, each referred to as a cycle (C₁, C₂, C_3, and C₄). At each cycle, a backcross was made with the respective A. hypogaea cultivar as the maternal parent and only lineages tracing back to a single hexaploid hybrid were used for RAPD analysis. Analysis of mapped, species-specific RAPD markers in BC₁F₁ to BC₁F₃ hybrids indicated that alien germplasm retention decreased every generation of inbreeding, especially in Argentine and in A. batizocoi crosses. A similar trend was also observed for every cycle in BC₁F₂ and BC₁F₃ families, possibly, due to the loss of alien chromosomes following selfing of hexaploids. RAPD marker analysis of 40–chromosome interspecific hybrid derivatives from the four crosses supported previous reports that reciprocal recombination and/or translocations are the predominant mechanisms for exchange of chromosomal segments. No evidence was found for preferential transfer of alien chromosomal regions to specific linkage groups. The implications for developing disease resistant peanut breeding lines are discussed in light of these findings.     

Thyroid receptor ligands. Part 5: novel bicyclic agonist ligands selective for the thyroid hormone receptor beta

Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.     

Genomic insights into acute inflammatory lung injury

Acute lung injury (ALI) is a devastating syndrome (usually associated with sepsis) that represents a major healthcare burden in the United States. We have focused our studies on unraveling the genetic underpinnings of this syndrome utilizing a candidate gene approach to identify novel genes for ALI susceptibility. Two novel genes identified by this approach include pre-B cell colony-enhancing factor (PBEF) and the gene for myosin light chain kinase (MLCK). PBEF protein levels were elevated in human bronchoalveolar lavage and serum samples from patients with ALI, and DNA sequencing identified two single nucleotide polymorphisms in the PBEF promoter (T-1001G, C-1543T) that were overrepresented in patients with sepsis-induced ALI. More recently, we found MLCK single polymorphisms and haplotypes to be associated with human ALI with unique variants observed in African-Americans with ALI. Thus genomic and genetic approaches represent powerful strategies in the identification of novel candidate genes and potential targets for ALI therapies.     

Three-dimensional mechanical properties of porcine coronary arteries: a validated two-layer model

The normal coronary artery consists of two mechanically distinct layers: intima-media and adventitia. The objective of this study is to establish a two-layer three-dimensional (3-D) stress-strain relation of porcine coronary arteries. Experimental measurements were made by a series of biaxial tests (inflation and axial extension) of intact coronary arteries and, subsequently, their corresponding intima-media or adventitia layer. The Fung-type exponential strain energy function was used to describe the 3-D strain-stress relation for each layer and the intact wall. A genetic algorithm was used to determine the material constants in the Fung-type constitutive equation by curve fitting the experimental data. Because one layer must be sacrificed before the other layer can be tested, the material property of the missing layer was computed from the material constants of the intact vessel and the tested layer. A total of 20 porcine hearts were used: one group of 10 hearts for the left anterior descending artery and another group of 10 hearts for the right coronary artery. Each group was further divided into two subgroups of five specimens tested for the intact wall and the intima-media layer and for the intact wall and the adventitia layer. Our results show statistically significant differences in the material properties of the two layers. The mathematical model was validated by experimental stress-strain data for individual layers. The validated 3-D constitutive model will serve as a foundation for formulation of layer-specific boundary value problems in coronary physiology and cardiology.     

Caspase-3 and caspase-6 in ductal breast carcinoma: a descriptive study

Caspases are the main point in the apoptotic process. We have collected some information from 210 cases of Ductal breast cancer (pT1 - pT2) such as tumour size, histological differentiation degree, lymph node status and tumor necrosis in the infiltrating component and we have evaluated the number of apoptotic cells or bodies by TUNEL technique as well as immunohistochemical studies to evaluate the expression of caspase 3 and caspase 6, and proliferation index. Our results show that lymph node status and cell atypism are independent prognostic factors for recurrence and mortality and only tumour size is an independent prognostic factor for recurrence. However, the apoptotic index and the immunohistochemical expression of caspases and cell proliferation index have not turned out to be independent prognostic factors neither for recurrence nor mortality. These results show that classic prognostic factors known until now are the most important factors to predict the evolution of the illness.     

Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.)

The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.     

Effects of eicosanoid biosynthesis inhibitors on the prophenoloxidase-activating system and microaggregation reactions in the hemolymph of Rhodnius prolixus infected with Trypanosoma rangeli

Investigations on the effects of eicosanoid biosynthesis inhibitors on the hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, parasitemia and mortality of Rhodnius prolixus infected with Trypanosoma rangeli were performed. Hemocoelic injection of live T. rangeli epimastigotes into fifth-instar larvae of R. prolixus that previously fed on blood containing an inhibitor of phospholipase A(2) (dexamethasone), a specific inhibitor of the cyclooxygenase pathway (indomethacin), and a non-selective lipoxygenase inhibitor (NDGA) (i) reduced the hemocyte microaggregation, (ii) attenuated the proPO system in the hemolymph and (iii) enhanced parasitemia and mortality induced by the parasite challenge in these insects. The effects obtained by dexamethasone administered orally were counteracted by inoculation of the insects with arachidonic acid. We suggest that the infectivity of T. rangeli can be increased by interference with the R. prolixus immune system. This is the first demonstration that the triatomine's immune responses to a parasite infection are modulated by a physiological system that includes eicosanoid biosynthesis.