Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Class VI Unconventional Myosin is Required for Spermatogenesis in Drosophila

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

A myosin from a higher plant has structural similarities to class V myosins

In plant cells, myosin is believed to be the molecular motor responsible for actin-based motility processes such as cytoplasmic streaming and directed vesicle transport. In an effort to characterize plant myosin, a cDNA encoding a myosin heavy chain was isolated from Arabidopsis thaliana. The predicted product of the MYA1 gene is 173 kDa and is structurally similar to the class V myosins. It is composed of the highly-conserved NH2-terminal "head" domain, a putative calmodulin-binding "neck" domain an alpha-helical coiled-coil domain, and a COOH-terminal domain. Northern blot analysis shows that the Arabidopsis MYA1 gene is expressed in all the major plant tissues (flower, leaf, root, and stem). We suggest that the MYA1 myosin may be involved in a general intracellular transport process in plant cells.

Molecular analysis of the myosin gene family in Arabidopsis thaliana

Myosin is believed to act as the molecular motor for many actin-based motility processes in eukaryotes. It is becoming apparent that a single species may possess multiple myosin isoforms, and at least seven distinct classes of myosin have been identified from studies of animals, fungi, and protozoans. The complexity of the myosin heavy-chain gene family in higher plants was investigated by isolating and characterizing myosin genomic and cDNA clones from Arabidopsis thaliana. Six myosin-like genes were identified from three polymerase chain reaction (PCR) products (PCR1, PCR11, PCR43) and three cDNA clones (ATM2, MYA2, MYA3). Sequence comparisons of the deduced head domains suggest that these myosins are members of two major classes. Analysis of the overall structure of the ATM2 and MYA2 myosins shows that they are similar to the previously-identified ATM1 and MYA1 myosins, respectively. The MYA3 appears to possess a novel tail domain, with five IQ repeats, a six-member imperfect repeat, and a segment of unique sequence. Northern blot analyses indicate that some of the Arabidopsis myosin genes are preferentially expressed in different plant organs. Combined with previous studies, these results show that the Arabidopsis genome contains at least eight myosin-like genes representing two distinct classes.

Preparation and characterization of TiO2/acid leached serpentinite tailings composites and their photocatalytic reduction of Chromium(VI)

Composite TiO2/acid leached serpentine tailings (AST) were synthesized through the hydrolysis–deposition method and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), energydispersive X-ray spectrometry (EDS), Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and surface area measurement (BET). The XRD analysis showed that TiO2 coated on the surface of acid leached serpentine tailings was mixed crystal phases of rutile and anatase, the grain size of which is 10–30 nm. SEM, TEM, and EDS analysis exhibited that nano-TiO2 particles were deposited on the surface and internal cavities of acid leaching serpentine tailings. The XPS and FT-IR analysis demonstrated that the coating process of TiO2 on AST was a physical adsorption process. The large specific surface area, porous structure, and plentiful surface hydroxyl group of TiO2/AST composite resulted in the high adsorption capacity of Cr(VI). The experimental results demonstrated that initial concentration of Cr(VI), the amount of the catalyst, and pH greatly influenced the removal efficiency of Cr(VI). The removal kinetics of Cr(VI) at a relative low initial concentration was fitted well with Langmuir–Hinshelwood kinetics model with R2 value of about unity. The asprepared composites exhibited strong adsorption and photocatalytic capacity for the removal of Cr(VI), and the possible photocatalytic reduction mechanism was studied. The photodecomposition of Cr(VI) was as high as 95% within 2 h, and the reusability of the photocatalysis was proven.

Fabrication of metal-nanoparticle-modified semiconducting copper--and silver--TCNQ materials as substrates for the reduction of chromium (VI) using thiosulfate ions at ambient temperature

The formation of readily recoverable and reusable organic semiconducting Cu- and AgTCNQ (TCNQ=7,7,8,8-tetracyanoquinodimethane) microstructures decorated with Pt and Pd metallic nanoparticles is described for the effective reduction of CrVI ions in aqueous solution at room temperature using both formic acid and an environmentally friendly thiosulfate reductant. The M-TCNQ (M=metal) materials were formed by electrocrystallisation onto a glassy carbon surface followed by galvanic replacement in the presence of H2PtCl6 or PdCl2 to form the composite material. It was found that loading of the surface with nanoparticles could easily be controlled by changing the metal salt concentration. Significantly, the M-TCNQ substrates facilitated the formation of well-isolated metal nanoparticles on their surfaces under appropriate galvanic replacement conditions. The semiconductor–metal nanoparticle combination was also found to be critical to the catalyst performance, wherein the best-performing material was CuTCNQ modified by well-isolated Pt nanoparticles with both formic acid and thiosulfate ions as the reductant.

Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala

Learning and memory depend on signaling mole- cules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the train- ing stimuli were presented in a non-associative manner. An- atomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically impli- cated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nu- cleus of the amygdala. When ML-7 was applied without as- sociative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the cir- cuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

A new approach to prepare ZVI and its application in removal of Cr(VI) from aqueous solution

Zero valent iron (ZVI) was prepared by reducing natural goethite (NG-ZVI) and synthetic goethite (SG-ZVI) in hydrogen at 550 °C. XRD, TEM, FESEM/EDS and specific surface area (SSA) and pore analyser were used to characterize goethites and reduced goethites. Both NG-ZVI and SG-ZVI with a size of nanoscale to several hundreds of nanometers were obtained by reducing goethites at 550 °C. The reductive capacity of the ZVIs was assessed by removal of Cr(VI) at ambient temperature in comparison with that of commercial iron powder (CIP). The effect of contact time, initial concentration and reaction temperature on Cr(VI) removal was investigated. Furthermore, the uptake mechanism was discussed according to isotherms, thermodynamic analysis and the results of XPS. The results showed that SG-ZVI had the best reductive capacity to Cr(VI) and reduced Cr(VI) to Cr(III). The results suggest that hydrogen reduction is a good approach to prepare ZVI and this type of ZVI is potentially useful in remediating heavy metals as a material of permeable reaction barrier.

Mutation of perinatal myosin heavy chain

Veugelers et al. (July 29 issue)1 report on patients with the trismus–pseudocamptodactyly syndrome as having a “Carney complex variant.” Among more than 500 patients with the Carney complex in our database, there are none with the trismus–pseudocamptodactyly syndrome.2,3...

Effect of Structural Modification on the Quantum-Size Effect in II-VI Semiconducting Nanocrystals

The electronic structure of group II-VI semiconductors in the stable wurtzite form is analyzed using state-of-the-art ab initio approaches to extract a simple and chemically transparent tight-binding model. This model can be used to understand the variation in the bandgap with size, for nanoclusters of these compounds. Results complement similar information already available for same systems in the zinc blende structure. A comparison with all available experimental data on quantum size effects in group II-VI semiconductor nanoclusters establishes a remarkable agreement between theory and experiment in both structure types, thereby verifying the predictive ability of our approach. The significant dependence of the quantum size effect on the structure type suggests that the experimental bandgap change at a given size compared to the bulk bandgap, may be used to indicate the structural form of the nanoclusters, particularly in the small size limit, where broadening of diffraction features often make it difficult to unambiguously determine the structure.

Theoretical studies on Beta-lactam antibiotics VI: Conformational analysis and structure-activity relationships of penicillin sulfoxides and cephalosporins

Conformational energy calculations were carried out on penicillin α-and Β-sulfoxides and δ2- and δ3-cephalosporins, in order to identify the structural features governing their biological activity. Results on penicillin Β-sulfoxide indicated that in its favoured conformation, the orientation of the aminoacyl group was different from the one required for biological activity. Penicillin α sulfoxide, like penicillin sulfide, favoured two conformations of nearly equal energies, but separated by a much higher energy barrier. The reduced activity of the sulfoxides despite the nonplanarity of their lactam peptide indicated that the orientations of the aminoacyl and carboxyl groups might also govern biological activity. δ3-cephalosporins favoured two conformations of nearly equal energies, whereas δ2-cephalosporins favoured only one conformation. The lactam peptide was moderately nonplanÄr in the former, but nearly planar in the latter. The differences in the.preferred orientations of the carboxyl group between penicillins and cephalosporins were correlated with the resistance of cephalosporins to penicillinases.

Unusual products of oxidation with Cr(VI) of methyl 5-methyl-7-methoxy-8-isopropyl-3,4-2-naphthoate

Abstract is not available.