Rhodium(I)-catalyzed 1,4-conjugate arylation toward ß-fluoroalkylated electron-deficient alkenes: a new entry to a construction of a tertiary carbon center possessing a fluoroalkyl group.

Treatment of ß-fluoroalkylated-α,ß-unsaturated ketones with 1.2 equiv. of various arylboronic acids in the presence of 5 mol% of [Rh(COD)(2)]BF(4) and 6 mol% of (S)-BINAP in toluene/H(2)O (v/v = 4/1) at the reflux temperature for 3 h gave the corresponding Michael adducts in high yields with over 90% enantioselectivity. Though other electron-deficient alkenes, such as vinylsulfone and vinylphosphonate, were found to be much less reactive in the rhodium-catalyzed conjugate addition with arylboronic acids, the reaction of various arylstannanes toward such electron-deficient alkenes took place very smoothly to afford the corresponding adducts in high yields.

The stability and aggregation properties of the GTPase domain from human SEPT4.

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTP. At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease.

Mammalian septins nomenclature.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.

Impaired expression of a human septin family gene Bradeion inhibits the growth and tumorigenesis of colorectal cancer in vitro and in vivo.

We have identified a novel human septin family gene Bradeion, which is specifically expressed in human colorectal cancer and malignant melanoma. In order to analyze the implications of tumor-specific gene expression, ribozymes and its derivatives were specifically designed and transfected into various colorectal adenocarcinoma cell lines for Bradeion inactivation. We constructed ribozyme expression plasmids controlled by a human tRNA(Val) promoter, and both hammerhead ribozyme and its allosteric derivative maxizyme were used for two different forms of Bradeion mRNA. The sequence-specific cleavage of Bradeion mRNA resulted in significant growth inhibition and G2 arrest in human cancer cell lines, detected by flow cytometry analysis. In addition, in vivo mice studies demonstrated marked tumor growth suppression by the Bradeion-specific ribozymes. Thus, the tumor-specific and selective marker Bradeion also provides valuable tools as a potential target for colorectal cancer therapy.

A sequential highly stereoselective hydroboration and Suzuki-Miyaura cross-coupling reaction of fluoroalkylated internal acetylenes: a practical one-pot synthesis of fluoroalkylated trisubstituted alkenes.

The one-pot synthesis of trisubstituted alkenes starting from fluoroalkylated internal alkynes was investigated. Hydroboration of the alkynes proceeded in a highly regio- and stereoselective manner to give the corresponding vinylboranes in excellent yields. Without isolation, treatment of the vinylboranes with various aryl halides under the Suzuki-Miyaura cross-coupling conditions gave the fluoroalkylated trisubstituted alkenes in high yields with complete retention of the olefinic geometry.

An intermediate structure in the thermal unfolding of the GTPase domain of human septin 4 (SEPT4/Bradeion-beta) forms amyloid-like filaments in vitro.

SEPT4 is a member of the mammalian septin family of GTPases. Mammalian septins are conserved proteins which form heteropolymers in vivo and which are implicated in a variety of cellular functions such as cytokinesis, exocytosis, and vesicle trafficking. However, their structural properties and modes of action are largely unknown. There is a limited, but as yet inconclusive, amount of experimental data suggesting that SEPT4 may accumulate in tau-based filamentous deposits and cytoplasmic inclusions in Alzheimer's and Parkinson's disease, respectively. Here we report an intermediate structure of the GTPase domain of human SEPT4 (SEPT4-G) during unfolding transitions induced by temperature. This partially unfolded intermediate, which is rich in beta-sheet and free of bound nucleotide, was plagued by irreversible aggregation. The aggregates have the ability to bind specific dyes such as Congo red and thioflavin-T, suggesting they are amyloid in nature. Under electron microscopy, fibers of variable diameter extending for several micrometers in length can be visualized. This is the first report of amyloid formation by a septin or domain thereof, and the capacity of SEPT4-G to form such fibrillar aggregates may shed some light on the current discussion concerning the formation of homo- and heteropolymers of septins in vitro.

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.

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.

Structure of the cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Cu,Zn superoxide dismutase (Cu,Zn SOD) is an essential enzyme for protecting cells from the toxic effects of reactive oxygen species. In humans, two distinct Cu,Zn SOD genes are located on chromosomes 4 and 21 and mutations in the latter have been associated with familial amyotrophic lateral sclerosis. Similarly, schistosomes (trematode parasites responsible for the chronically debilitating disease schistosomiasis) also produce two distinct Cu,Zn SODs, in this case one cytosolic and one bearing a signal peptide. The crystal structure of the cytosolic form of the enzyme from the human trematode Schistosoma mansoni (SmCtSOD) was solved and refined to a resolution of 2.2 A (space group P2(1)2(1)2(1), R = 17.6% and R(free) = 24.1%) and 1.55 A (space group P2(1), R = 15.7% and R(free) = 17.1%). This is the first report of a crystal structure of a Cu,Zn superoxide dismutase derived from a human parasite. Alternate positions for the catalytic copper and its water ligand were refined for the 1.55 A SmCtSOD model, but the most interesting structural differences between SmCtSOD and the human homologue reside in the loops used for electrostatic guidance of the substrate to the enzyme active site.

Rapid and quantitative detection of human septin family Bradeion as a practical diagnostic method of colorectal and urologic cancers.

BACKGROUND: Malignant tumor progression is a complex and multi-gene event which can not be easily detected or predicted. The detection of malignant cells using marker genes is hampered by the fact that these markers are only expressed by certain malignancies or lack sensitivity and/or specificity. We have reported a human septin family gene Bradeion, which shows strong cancer-specific expression in colorectal and urologic cancers as a result of carcinogenesis. MATERIAL/METHODS: Diagnostic efficacy and validity of Bradeion gene expression were tested by two independent systems, one is a protein detection method using monoclonal antibody based immuno-chromatographic membrane strip tests (a nitrocellulose test strip assay), and another is a gene expression detection method, quantitative RT-PCR. The technology has been established using Bradeion fusion proteins, in vitro cultivated human cancer cell lines, and also patients' test samples with controls. RESULTS: Bradeion test strip by combination with two monoclonal antibodies are valid for the detection of 1 ng/ml Bradeion, and successfully applied for patient urine samples with no false-positive results. Positive detection rates were over 70% of the patient urine samples so far tested (prostate cancer, renal cell carcinoma, and bladder cancer) in 15 to 30 minutes. Quantitative RT-PCR resulted in significantly high copy numbers of 0.4-3.0-3.0 x 10(5) per microg total RNA in patients' tissue samples, whereas those from normal tissue or other cancers found negative. CONCLUSIONS: The present study introduces the practical diagnostic methods using a disease-specific molecular marker, which provides safe, economical, and rapid clinical screening of cancer.

The serodiagnosis of amoebiasis by enzyme-linked immunosorbent assay

We attempt to detect antibodies against Entamoeba histolytica in sera obtained from both healthy individuals and hospitalized patients by an enzyme-linked immunosorbent assay (ELISA). The limiting value for ELISA-positive was established on the basis of serologic results of 1869 healthy persons living in a non-endemic area (Japan)> The ELISA then was applied to 61 patients in 5 hospitals in Myanmar who were suspected of having amoebiasis. The ELISA results were compared with those of stool examinations, and evaluated for sensitivity. Using the llimiting value for the ELISA, there was an 83 Percent agreement of positivity between the ELISA tests and microscopic examinations. Specific anti-E-histolytica IgM was observed in the sera of 44 patients; IgG in all of the 61 patients. Since high titers of specific IgM were not observed in both stool-positive and stool-negative patients, we judged that patients with early stages of amoebic infections were not included in this population. In our ELISA, the sera of the patients did not exhibit crosss-reactions betweeen antigens prepared from the HK-9 strain of E. histolytica and antigens prepared from Trichomonas vaginalis and Giardia lamblia. On the basis of these results, we suggest that it is necessary to do two or more different examinations in order to make a definite diagnosis of amoebic infection.

Detection of Entamoeba histolytica antigen in stool samples by enzyme-linked immunosorbent assay using monoclonal antibodies

An enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, was applied for the detection of Entamoeba histolytica antigen in stool samples obtained from 148 patients with gastrointestinal symptoms, and the results are compared with microscopic findings. Ninety nine positives by microscopy generally had high ELISA OD values. Ninety one stool samples of asymptomatic cyst passers were also investigated by ELISA, and most were found to be positive. Although false positives were observed in both symptomatic and asymptomatic cases, the ELISA appears to be useful for the detection of amoebic antigen(s). However, our results suggest that both immunological methods and microscopic examination are needed for an accurate diagnosis of intestinal amoebiasis.

Proceedings of the schistosome genome project

"The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery. Clearly, the adaptation of parasites to their different host species, and to the different environomental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin. The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories. Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm an ZW for a female), of a haploid genome size of 2.7X10 8 base pairs (Simpson et al. 1982). Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165Mb), and contain functional genes with a high level of homology to the host mammalian genes. Here we summarize the current progress in the schistosome genome project, the information of 3.047 transcribed genes (Expresses Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes. The schistosome genome project will further identify and charaterize the key molecules that are responsable for host-parasite adaptation, i.e., successful growth developement, maturation and reproduction of the parasite within its host in the near future.