Microbial communities show parallels at sites with distinct litter and soil characteristics

Plant and microbial community composition in connection with soil chemistry determines soil nutrient cycling. The study aimed at demonstrating links between plant and microbial communities and soil chemistry occurring among and within four sites: two pine forests with contrasting soil pH and two grasslands of dissimilar soil chemistry and vegetation. Soil was characterized by C and N content, particle size, and profiles of low-molecular-weight compounds determined by high-performance liquid chromatography (HPLC) of soil extracts. Bacterial and actinobacterial community composition was assessed by terminal restriction fragment length polymorphism (T-RFLP) and cloning followed by sequencing. Abundances of bacteria, fungi, and actinobacteria were determined by quantitative PCR. In addition, a pool of secondary metabolites was estimated by erm resistance genes coding for rRNA methyltransferases. The sites were characterized by a stable proportion of C/N within each site, while on a larger scale, the grasslands had a significantly lower C/N ratio than the forests. A Spearman's test showed that soil pH was correlated with bacterial community composition not only among sites but also within each site. Bacterial, actinobacterial, and fungal abundances were related to carbon sources while T-RFLP-assessed microbial community composition was correlated with the chemical environment represented by HPLC profiles. Actinobacteria community composition was the only studied microbial characteristic correlated to all measured factors. It was concluded that the microbial communities of our sites were influenced primarily not only by soil abiotic characteristics but also by dominant litter quality, particularly, by percentage of recalcitrant compounds.     

Caenorhabditis elegans expresses three functional profilins in a tissue-specific manner

Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L-proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell-cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression.     

Structural and functional characteristics of the CD3 (T3) molecular complex on human thymocytes

The CD3 (T3) molecular complex is noncovalently associated with the antigen receptor molecule on T cells. The mitogenic properties of anti-CD3 antibodies have suggested that this complex may be the transducer of the antigenic signal to the intracellular environment. In the present investigation, we studied some of the structural and functional characteristics of the CD3 complex on human thymocytes. In 11 specimens tested, we found that anti-CD3 antibodies react with 50 to 76% of the thymocytes. Two-color immunofluorescence analysis revealed that the majority (greater than 50%) of thymocytes express both CD3 and CD1 on their surfaces. The latter is a marker of immature thymocytes. However, a distinct subpopulation comprising 13 to 19% of the total cells displays only CD3, while an approximately equal percentage of cells expresses only CD1. The mitogenic potential of anti-CD3 antibodies on peripheral T cells is dependent on the presence of monocytes. Anti-CD3 antibodies by themselves cannot activate thymocytes, indicating that functionally active monocytes are absent from the thymocyte population. Even the addition of peripheral monocytes does not allow a response of thymocytes to anti-CD3 antibodies. However, when the anti-CD3 antibody 64.1 is added in the presence of exogenous rIL 2, a strong antibody and lymphokine dose-dependent response ensues. Only CD1- CD3+ thymocytes are stimulated by the addition of antibody and IL 2. The mere expression of CD3 on the CD1+ CD3+ subpopulation of thymocytes apparently is not sufficient to render the cells responsive to the signals of anti-CD3 and IL 2.     

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.     

Cyclin D1 and cyclin D3 show divergent responses to distinct mitogenic stimulation

D‐type cyclins predominantly regulate progression through the cell cycle by their interactions with cyclin‐dependent kinases (cdks). Here, we show that stimulating mitogenesis of Swiss 3T3 cells with phorbol esters or forskolin can induce divergent responses in the expression levels, localization and activation state of cyclin D1 and cyclin D3. Phorbol ester‐mediated protein kinase C stimulation induces S phase entry which is dependent on MAPK activation and increases the levels and activation of cyclin D1, whereas forskolin‐mediated cAMP‐dependent protein kinase A stimulation induces mitogenesis that is independent of MAPK, but dependent upon mTor and specifically increases the level and activation of cyclin D3. These findings uncover additional levels of complexity in the regulation of the cell cycle at the level of the D‐type cyclins and thus may have important therapeutic implications in cancers where specific D‐cyclins are overexpressed. J. Cell. Physiol. 225: 638–645, 2010. © 2010 Wiley‐Liss, Inc.     

Structural and biochemical properties show ARL3-GDP as a distinct GTP binding protein

BACKGROUND: Based on sequence similarities, Arf-like (ARL) proteins have been assigned to the Arf subfamily of the superfamily of Ras-related GTP binding proteins. They have been identified in several isoforms in a wide variety of species. Their cellular function is unclear, but they are proposed to regulate intracellular transport. RESULTS: The 1.7 A crystal structure of murine ARL3-GDP provides a first insight into the structural features of this subgroup of Ar proteins. The N-terminal extension of ARL3 folds into an elongated loop region that is hydrophobically anchored onto the surface by burying 1440 A2. The features observed suggest that ARL3 releases its N terminus and undergoes a beta sheet register shift upon the binding of GTP. The structure and kinetic experiments with fluorescent mGDP demonstrate that tight GDP (but not GTP) binding is achieved in the absence of a magnesium ion. This is due to a lysine residue in the active site, close to the canonical Mg2+ site found in other GTP binding proteins. This is a distinct feature separating ARL2 and ARL3 from Arf proteins. CONCLUSION: The disturbed magnesium binding site and the independence of GDP coordination from the presence of Mg2+ separate ARL2 and ARL3 from Arf proteins. The D sheet register shift, which is similar to that of Arf, that is observed in the present structure, along with the postulated release of the N-terminal extension and the concomitant exposure of a patch of conserved hydrophobic residues in this region suggest that ARL proteins might be localized to target membranes upon exchange of GDP to GTP. Contrary to the situation in Arf, however, the conformational change to ARL-GTP does not require the presence of membranes and might thus be energetically unfavored. Together with the very low affinity described for the interaction of ARL3 with Mg-GTP, this suggests that ARL protein activation requires the presence of effectors stabilizing the GTP coordination rather than guanine nucleotide exchange factors (GEFs).     

Differentiation of the acrosomal complex in ostrich (Struthio camelus) spermatids

The acrosomal complex of ostrich sperm consists of a small, cone-shaped acrosome and a slender, cylindrical perforatorium housed within a deep endonuclear canal. The perforatorium is almost exclusively endonuclear in location and is only covered by the acrosome at its point of origin in the apical subacrosomal space. The development of the acrosome is generally similar to that described in other non-passerine birds. Small proacrosomal granules (vesicles) emanating from the Golgi apparatus coalesce to form a large, membrane-bound acrosomal vesicle filled with homogeneous, electron-dense material. The acrosomal vesicle attaches to the nucleus via a shallow depression and subsequently collapses to form the typical cap-like acrosome of non-passerine birds. In ostrich spermatids the endonuclear canal becomes obvious when the collapsed acrosomal vesicle has assumed a dumbbell-shaped appearance. The perforatorium, which originates from moderately electron-dense material contained within the apical subacrosomal space, expands within the deepening endonuclear canal. The material of the perforatorium does not originate in the form of an obvious granule as in chicken and budgerigar spermatids. Indications are that in ostrich spermatids the developing acrosome plays a role in the shaping of the tip of the nucleus. The perforatorium, however, appears to represent a residual structure that has no specifically identified function. © 1996 Wiley-Liss, Inc.     

Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.     

Mutational analysis reveals distinct features of the Nox4-p22 phox complex

The integral membrane protein p22(phox) forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22(phox) constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22(phox) essential for formation and localization of the Nox4-p22(phox) complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22(phox)-deficient and promotes cell surface expression of the Nox4-p22(phox) heterodimer. Expression of p22(phox) truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22(phox) interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22(phox), which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22(phox) mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22(phox) association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22(phox) subunit with Nox4, a feature which could provide the basis for selective inhibitor development.     

Expression and functional roles of the two distinct NDH-1 complexes and the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 in Synechocystis sp PCC 6803

To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DeltaNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the DeltaNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (DeltaNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in DeltaNdhD1/D2 and M55. In contrast with the wild type and DeltaNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the DeltaNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3- transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

New generation CPPs show distinct selectivity for cancer and noncancer cells

In the last three decades, many new cell-penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini-crotamine and mini-maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV-TAT(47-57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N-terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG-peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image-guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.     

On the origin and evolution of vertebrate and viral profilins

The three dimensional structures of profilins from invertebrates and vertebrates are remarkably similar despite low sequence similarity. Their evolutionary relationship remains thus enigmatic. A phylogenetic analysis of profilins from Deuterostoma indicates that profilin III and IV isoforms each form distinct groups. Profilin IV is most related to invertebrate profilins and originated prior to vertebrate evolution whereas separation of profilin I, II and III isoforms occurred early in vertebrate evolution. Viral profilins are most similar to profilin III. In silico analysis of representative profilin gene structures corroborates the phylogenetic result and we discuss this in terms of biochemical differences.     

Ultrastructural labeling of the chromatoid body and the centriole-associated body using the DNase-gold colloidal complex in monkey spermatids

In Monkey spermatids at different steps of spermiogenesis, the use of DNase-gold complex showed, at the ultrastructural level, a labeling over the chromatin and concomitantly over the chromatoid body, centriole associated body and annular chromatoid body. The results obtained with the DNase-gold complex containing either DNA or actin led to discuss the nature of the substances revealed by the labeling in the cytoplasmic structures.