Sarcoglycan subcomplex expression in normal human smooth muscle.

The sarcoglycan complex (SGC) is a multimember transmembrane complex interacting with other members of the dystrophin-glycoprotein complex (DGC) to provide a mechanosignaling connection from the cytoskeleton to the extracellular matrix. The SGC consists of four proteins (alpha, beta, gamma, and delta). A fifth sarcoglycan subunit, epsilon-sarcoglycan, shows a wider tissue distribution. Recently, a novel sarcoglycan, the zeta-sarcoglycan, has been identified. All reports about the structure of SGC showed a common assumption of a tetrameric arrangement of sarcoglycans. Addressing this issue, our immunofluorescence and molecular results showed, for the first time, that all sarcoglycans are always detectable in all observed samples. Therefore, one intriguing possibility is the existence of a pentameric or hexameric complex considering zeta-sarcoglycan of SGC, which could present a higher or lower expression of a single sarcoglycan in conformity with muscle type--skeletal, cardiac, or smooth--or also in conformity with the origin of smooth muscle.     

Isolation and characterization of a new mutant allele of <Emphasis Type=Italic>brachytic 2</Emphasis> maize gene

More than 40 monogenic dwarfing mutants have been described in maize; however, the majority of these lead to great reductions in grain yield and, consequently, they have not been used to enhance crop yield in germplasm that is sensible to lodging. An exception in terms of commercial value is the maize mutant brachytic 2 (br2). Br2 gene was cloned in 2003, and it encodes for a protein most probably involved in auxin polar transport. We have isolated a new brachytic mutation that is allelic to the br2 locus and denoted this novel mutant as br2–23. Characterization of this mutant revealed that the br2 mutation modified not only the length of the internodes, as previously reported, but the structure of the leaves as well. Br2–23/br2–23 heterozygotes have a useful intermediate phenotype in terms of plant height, ear height and leaf angle, suggesting a possible utilization of this effect in developing new hybrids. This mutant also appears to be an useful tool by which to study the switch points of the complex developmental program determining maize plant height and architecture.     

DC-EPG assisted comparison of European spittlebugs and sharpshooters feeding behaviour on grapevine

Xylem-feeding is apparently the only requirement making an insect a competent vector of the bacterium Xylella fastidiosa, an organism responsible for the devastation of the Southern Italian olive forest and nowadays considered one of the most feared threats to agriculture and landscape in Europe, including vineyards. Here, we used the direct current-electrical penetration graph (DC-EPG) technique to compare and describe the feeding behaviour on grapevine of four xylem-feeding species considered candidate vectors of X. fastidiosa widespread in Europe, namely two spittlebugs (the meadow spittlebug Philaenus spumarius and the spittlebug Neophilaenus campestris) and two sharpshooter leafhoppers (the rhododendron leafhopper Graphocephala fennahi and the green leafhopper Cicadella viridis). We created a standard for the analysis of EPG waveforms recorded with a DC-EPG device, describing feeding activities performed by these insects from stylet insertion into the plant to withdrawal. This standard, along with freely available software, has been developed to harmonize the calculation of feeding behavioural parameters in xylem-feeders. The most relevant differences between the two vector taxa were the probing frequency and the dynamics of xylem ingestion. Sharpshooters tended to perform significantly more probes than spittlebugs. In contrast, the latter spent longer times in low-frequency xylem ingestion, characterized by scattered contractions of the cibarial dilator muscle interspersed with periods of pump inactivity. Cicadella viridis was the species displaying the highest frequency of the electrical pattern found to be associated with X. fastidiosa inoculation in spittlebugs (Xe). Feeding behavioural data presented here represent an important step forward for deepening our knowledge of xylem-sap feeding insects' interaction with both the host plants and the bacterium they transmit.     

Gene and protein expressions in human cord blood cells after exposure to acrylonitrile

Acrylonitrile is a very high volume industrial chemical used primarily in the manufacture of plastics and rubber, which displays a pronounced acute toxicity and may be carcinogenic. The damage to the hematopoietic function by acrylonitrile may result from interference with cytokine production and cytokine receptor binding. Our present data show that acrylonitrile modulates the expression of some genes implicated in cell differentiation, cell-cycle progression, and clonogenic potential of human cord blood cells. A macroarray hybridization analysis showed that expression of the CXCR4, MCP-1, and MRP8 genes was modified by acrylonitrile exposure. Moreover, the acrylonitrile cell target seems to be the myeloid compartment, as assessed by a CFU-GM assay. In particular, the downregulation of CXCR4, MCP1, and MRP8 can be responsible for the observed reduction of cell proliferation and clonogenic capability of CFU-GM precursors. A Western blot assay showed an acrylonitrile-dependent induction of Bax, while Bcl-2 expression changed only after 48 h of chemical exposure. Bax was overexpressed in respect to Bcl-2, and this fact can be responsible for the induction in cell death after 24 h of treatment. C-fos and c-jun were also downregulated after 24 h and 6 h of treatment, respectively.     

The challenge of brain lipidomics

After many years backstage, lipids have made a come back in the limelight of neuroscience. This renewed excitement was sparked by a series of convergent discoveries in the fields of neural development, synaptic physiology and receptor pharmacology, which have begun to reveal the roles played by lipid messengers and their receptors in brain function. Such roles extend from the development of the neocortex to the processing of complex behaviors, encompassing a territory as vast as those traditionally assigned to growth factors, neurotransmitters and neuropeptides. Along with these basic discoveries, technical advances have simplified the identification and quantification of neural lipids, achieving a degree of sensitivity and selectivity that was unthinkable only 10 years ago. Thanks to this progress, we can now resolve complex mixtures of lipid molecules and quantify each of their components, which are often present in tissues at vanishingly low concentrations. In this review, I outline several key features of brain lipid signaling and discuss the opportunities and challenges that such features impose on future lipidomic approaches.     

Report on antibodies submitted to the stromal cell section of HLDA8

The paradigm for tissue specific homing of leukocytes is the "area code" hypothesis, which predicts that a specific combination of adhesive interactions and chemokine signals from the endothelium directs leukocyte migration into specific tissue sites. This area code hypothesis has been supported by studies from previous HLDA workshops where endothelial specific cell antigens have been studied. Similarly, a clear haematopoietic "stem cell code" comprising the chemokine SDF-1 (CXCL12) and the adhesion receptor VCAM-1 (CD106) has been shown to contribute to the stem cell niche within bone marrow [K. Tokoyoda, T. Egawa, T. Sugiyama, B.I. Chai, T. Nagasawa, Cellular niches controlling B lymphocyte behaviour within bone marrow during development, Immunity 20 (2004) 707-718]. HLDA 7 included a section devoted to stem cell antigens, which began to define additional antigens important in these processes. During the course of HLDA 8 we have extended these observations to determine whether a more global stromal address code defined by fibroblasts, exists in variety of different tissues [G. Parsonage, A.D. Filer, O. Haworth, G.B. Nash, G.E. Rainger, M. Salmon, C.D. Buckley, A stromal area postcode defined by fibroblasts, Trends Immunol. 26 (2005) 150-156]. The stromal cell section in HLDA 8 was designed to complement the malignant cell, endothelial cell, and stem cell/progenitor cell sections. Seven new CD numbers were assigned to antibodies included in this section at the HLDA 8 Workshop meeting held during December 2004.     

Depression by relaxin of neurally induced contractile responses in the mouse gastric fundus

The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.     

Crystal structure of cytoglobin: the fourth globin type discovered in man displays heme hexa-coordination

Cytoglobin is a recently discovered hemeprotein belonging to the globin superfamily together with hemoglobin, myoglobin and neuroglobin. Although distributed in almost all human tissues, cytoglobin has not been ascribed a specific function. Human cytoglobin is composed of 190 amino acid residues. Sequence alignments show that a protein core region (about 150 residues) is structurally related to hemoglobin and myoglobin, being complemented by about 20 extra residues both on the N and C termini. In the absence of exogenous ligands (e.g. O2), the cytoglobin distal HisE7 residue is coordinated to the heme Fe atom, thus decreasing the ligand affinity. The crystal structure of human cytoglobin (2.1 A resolution, 21.3% R-factor) highlights a three-over-three alpha-helical globin fold, covering residues 18-171; the 1-17 N-terminal, and the 172-190 C-terminal residue segments are disordered in both molecules of the crystal asymmetric unit. Heme hexa-coordination is evident in one of the two cytoglobin chains, whereas alternate conformation for the heme distal region, achieving partial heme penta-coordination, is observed in the other. Human cytoglobin displays a large apolar protein matrix cavity, next to the heme, not related to the myoglobin cavities recognized as temporary ligand docking stations. The cavity, which may provide a heme ligand diffusion pathway, is connected to the external space through a narrow tunnel nestled between the globin G and H helices.     

Prefibrillar amyloid protein aggregates share common features of cytotoxicity

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.     

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.