Multifunctional roles for the PH domain of Dbs in regulating Rho GTPase activation

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.     

A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.     

中国的炭疽杆菌DNA分型及其地理分布

炭疽广泛分布于中国各地,特别是西部地区,并经常造成人畜疾病,在一项合作研究中,用多位点VNTR分析（MLVA）对从1952-1998年自中国主要地理流行区域分离的病人,病畜和土壤等来源的炭疽杆菌进行了基因分型,MLVA分析结果揭示了21种新的基因型,其等位基因组合在以前世界范围分离物的研究中未曾发现,此外,分离物的分群显示,A3b组是地理上最广泛分布的基因组,说明该组可能是中国的“地方流行株”。而来自古丝绸之路重要贸易中心新疆的大量分离株其基因型特别分散。     

Systematics of genus Gnomoniopsis (Gnomoniaceae, Diaporthales) based on a three gene phylogeny, host associations and morphology

Species of Gnomoniopsis are leaf- and stem-inhabiting pyrenomycetes that infect plants in Fagaceae, Onagraceae and Rosaceae. Morphology and analyses of DNA sequences from three ribosomal DNA and protein coding regions, namely β-tubulin, translation elongation factor 1α (tef-1α) and the ITS region including ITS1, 5.8S rDNA and ITS2, were used to define species in Gnomoniopsis. Secondary structural alignment of the ITS region across four genera in Gnomoniaceae was used to increase the potential number of homologous positions in the ITS alignment. Ascospore isolates were grown from newly collected specimens. Type specimens were compared with these specimens to determine their identity. In this paper a recent concept of Gnomoniopsis is confirmed with phylogenetic resolution of additional species. Four new combinations and one new species are proposed. Nine species are described and illustrated, and a key is provided to the 13 species currently recognized in Gnomoniopsis.     

Tubeufia asiana, the teleomorph of Aquaphila albicans in the tubeufiaceae, pleosporales, based on cultural and molecular data

The teleomorph of Aquaphila albicans was discovered on submerged wood collected in Thailand. Its black, soft-textured, setose ascomata, bitunicate asci and hyaline to pale brown, multiseptate ascospores indicated an affinity to Tubeufiaceae (Dothideomycetes). After morphological or molecular comparisons with related species in Tubeufia, Acanthostigma and Taphrophila, it is described and illustrated as a new species, T. asiana Sivichai & K.M. Tsui, sp. nov. Finding this Tubeufia teleomorph was surprising, given the falcate conidia of its A. albicans anamorph, which superficially resemble the conidia of Fusarium and not the coiled, helicosporous conidia of other species in Tubeufiaceae. We assessed the phylogenetic relationships of A. albicans-T. asiana with ribosomal sequences from SSU and ITS and partial LSU regions by parsimony and Bayesian analysis. An initial set of 40 taxa representing a wide range of ascomycete families and their SSU sequences from GenBank showed A. albicans-T. asiana to be nested within the Tubeufiaceae with 100% bootstrap support. Their placement was inferred with ITS and partial LSU ribosomal sequences. The nearly identical ITS sequences of two isolates of A. albicans and one isolate of Tubeufia asiana united these fungi as a monophyletic group with 100% bootstrap support and further nested them, with 88% bootstrap support, in a clade containing Helicoon gigantisporum and Helicoma chlamydosporum. This is the first molecular phylogenetic study to place a nonhelicosporous species within the Tubeufiaceae and to show that helical conidia were lost at least once within the family.     

Sex-based differences in myocardial contractile reserve

Recent studies have identified sex differences in heart function that may affect the risk of developing heart failure. We hypothesized that there are fundamental differences in calcium (Ca) regulation in cardiac myocytes of males and premenopausal females. Isometric force transients (n = 45) were measured at various stimulation frequencies to define the force frequency responses (FFR) (0.5, 1.0, 1.5, and 2.0 Hz) during either changes in bath Ca ([Ca]o) (1.0, 1.75, 3.5, and 7.0 mM) or length-tension (20, 40, 60, 80, and 100% L(max)) in right ventricle trabeculae from normal male (MT) and premenopausal female (FT) cats. Force-Ca measurements were also obtained in chemically skinned trabeculae. Under basal conditions (0.5 Hz, 1.75 mM Ca, 80% L(max)) both MT and FT achieved similar developed forces (DF) (MT 11 +/- 1, FT = 10 +/- 1 mN/mm2). At low rates and lengths, there is no sex difference. At higher preloads and rates, there is a separation in DF in MT and FT. At basal [Ca]o both MT and FT exhibited positive FFR (2.0 Hz, 1.75 mM Ca: MT 38 +/- 3, FT 21 +/- 4 mN/mm2); however, at higher [Ca]o, MT achieved greater DF (2.0 Hz, 7.0 mM Ca: MT 40 +/- 3 and FT = 24 +/- 4 mN/mm2). We detected no sex difference in myofilament Ca sensitivity at a sarcomere length of 2.1 mum. However, rapid cooling contractures indicated greater sarcoplasmic reticulum (SR) Ca load in MT at higher frequencies. Despite virtually identical contractile performance under basal conditions, significant sex differences emerge under conditions of increased physiological stress. Given the lack of sex differences in myofilament Ca sensitivity, these studies suggest fundamental sex differences in cellular Ca regulation to achieve contractile reserve, with myocardium from males exhibiting higher SR Ca load.     

The type species of Apiognomonia, A. veneta, with its Discula anamorph is distinct from A. errabunda

Species of Apiognomonia with their Discula anamorphic states in the Gnomoniaceae, Diaporthales, are known throughout the temperate Northern Hemisphere and cause diseases such as sycamore or plane tree anthracnose. The genus Apiognomonia was described based on A. veneta as the type species; however, there has been disagreement about whether or not A. veneta is a synonym of A. errabunda. Using morphological, ecological, and DNA sequence data we conclude that A. errabunda and A. veneta are different species, although very closely related; thus, A. veneta is the correct name for the type species of Apiognomonia. This conclusion is based on a combined analysis of sequences from the ITS regions of nuclear rDNA for 51 isolates from host plants of eight genera and intron regions from actin, calmodulin and translation elongation factor 1-alpha for over 25 isolates. The type species of the genus Discula is D. nervisequa, the earliest available epithet for D. platani, the lectotype of Discula. D. nervisequa is the anamorph of A. veneta. Based on an examination of the type specimen, we determined that the commonly used name for the anamorph of A. errabunda, D. umbrinella, refers to another species. A. veneta and A. errabunda including their anamorphs are described and illustrated. An account of all synonyms and excluded synonyms is presented.     

Foraging habits in a generalist predator: Sex and age influence habitat selection and resource use among bottlenose dolphins (Tursiops truncatus)

This study examines resource use (diet, habitat use, and trophic level) within and among demographic groups (males, females, and juveniles) of bottlenose dolphins (Tursiops truncatus). We analyzed the δ¹³C and δ¹⁵N values of 15 prey species constituting 84% of the species found in stomach contents. We used these data to establish a trophic enrichment factor (TEF) to inform dietary analysis using a Bayesian isotope mixing model. We document a TEF of 0‰ and 2.0‰ for δ¹³C and δ¹⁵N, respectively. The dietary results showed that all demographic groups relied heavily on low trophic level seagrass‐associated prey. Bayesian standard ellipse areas (SEA_b) were calculated to assess diversity in resource use. The SEA_b of females was nearly four times larger than that of males indicating varied resource use, likely a consequence of small home ranges and habitat specialization. Juveniles possessed an intermediate SEA_b, generally feeding at a lower trophic level compared to females, potentially an effect of natal philopatry and immature foraging skills. The small SEA_b of males reflects a high degree of specialization on seagrass associated prey. Patterns in resource use by the demographic groups are likely linked to differences in the relative importance of social and ecological factors.     

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: a template-based approach--part 2

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.     

MicroRNAs in systemic rheumatic diseases

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.