Seimatoantlerium tepuiense gen. nov., a unique epiphytic fungus producing taxol from the Venezuelan Guyana.

Seimatoantlerium gen. nov., type species, S. tepuiense sp. nov. is proposed for an acervular fungus producing 4-septate, holoblastic conidia with 6-8 unbranched, apical appendages that dehisce as an appendage apparatus and also commonly possessing one or two exogenous basal appendages as well as a pedicel. It is compared with Seimatosporium, Seimatosporiopsis, and other genera. It is epiphytic on Maguireothamnus speciosus, a rubiaceous plant endemic to the tepuis of southeastern Venezuela. It produces the anti-oomycetous anticancer compound, taxol, as shown by immunological and spectroscopic methods. Taxol production is discussed relative to the ability of this fungus to exist in an extremely moist ecosystem, as well as to its relationship to other plant associated fungi.     

Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets

Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.     

A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.     

Dynamic nature of a wheat centromere with a functional gene

Centromeric regions of higher eukaryotes are comprised mainly of tandem and non-tandem repeat sequences with variable copy number, spacing, order and orientation; are heterochromatic in nature, and are believed to be devoid of actively transcribing genes. Here, we report an actively transcribing wheat homolog of HSP70 gene that maps in the functional wheat centromere, and copy number of which seems to change in response to centromeric breaks. The HSP70 gene physically maps on the short arm of chromosomes 1A and 1D of Chinese Spring (CS) and 1R of rye. Whereas, on chromosome 1B in both ‘CS’ and Pavon background, the gene maps in the functional centromere as evident from its presence in both cytologically confirmed true ditelosomic lines Dt1BS and Dt1BL. Sequence comparison of 11 ESTs showed three sequence patterns suggesting that all three homoeologous copies of the gene are expressing. The cDNA-single stranded conformation polymorphism analysis confirmed expression of the ‘CS’ 1B copy of the gene. Observed in two independently developed Dt1BL lines, the 1B copy number of the gene showed three to fivefold increase in response to chromosomal breaks around the centromere. Putative gene duplications seem to involve large chromosomal segments as only one of the ten restriction enzymes used for DNA gel-blot analysis showed unique extra fragment band in the Dt1BL line. Further investigations are warranted to uncover the nature and mechanism of these duplications.     

A hydrogen bond regulates slow motions in ubiquitin by modulating a β-turn flip

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions—the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.     

Two distinct pathways for cyclooxygenase-2 protein degradation

Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.     

GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein

Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson''s disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3β because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3β. GSK-3β-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3β to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3β-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3β-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3β directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3β. Together, these data establish a novel upstream role for GSK-3β as one of several kinases associated with PTMs of key proteins known to be causal in PD.After Alzheimer''s disease (AD), Parkinson''s disease (PD) is the second most prevalent neurodegenerative disease, characterized by selective loss of TH+ DA-neurons of substantia nigra (SN) with diminished production of dopamine (DA).¹ Genome-wide studies have identified SNCA and MAPT, genes encoding α-synuclein (α-Syn) and Tau, respectively, as having strong association to the genesis of PD.^{2, 3, 4} Although the precise etiology of PD remains a mystery, SNCA amplifications and mutations directly link α-Syn dysfunction to disease causation,^{5, 6} firmly establishing a role for α-Syn in sporadic and familial PD, respectively. α-Syn can be phosphorylated at several sites,⁷ and the predominance of α-Syn phosphorylated at serine 129 (S129) in Lewy bodies⁸ suggests its phosphorylation status at S129 has an important pathological role. Various PD models have shown that phosphorylation at S219 enhanced α-syn toxicity resulting in accelerated motor abnormalities and loss of DA-neurons.^{9, 10}Fewer studies have examined the role of Tau (or p-Tau) in PD, but interest in the field has grown since completion of several genome-wide association studies. p-Tau has been found to colocalize with α-Syn in tissue from sporadic PD and dementia with Lewy bodies.¹¹ We^{12, 13} and others^14,15 have also identified p-Tau in different brain regions of PD, dementia with Lewy bodies, and AD. High levels of p-Tau have also been observed in vivo in several toxin^{16, 17, 18} and transgenic α-Syn models of PD,^19,20 suggesting that p-Tau may be an important common factor in the neurodegeneration of not only tauopathies but also of synucleinopathies, such as PD.^{21, 22, 23, 24} Most studies to date have focused on the formation and accumulation of Tau and p-Tau in idiopathic PD. Yet several studies have provided evidence that leucine-rich repeat kinase-2 (LRRK2), a kinase, that when mutated is involved in familial forms of PD, can directly interact with, and activate GSK-3β, resulting in increased p-TAU formation.^25,26Among the kinases known to hyperphosphorylate Tau, glycogen synthase kinase-3β (GSK-3β) may be the most important given its ability to phosphorylate Tau at the majority of its serine/threonine sites that cause associated toxicities in AD.^27,28 The importance of GSK-3β is illustrated in that it is embryonically lethal when knocked out in mice. Regulation of GSK-3β is tightly controlled through a series of direct and indirect measures. Direct regulation occurs through autophosphorylation at Tyr216,^29,30 resulting in a kinase-active form, p-GSK-3β-Y216, whereas phosphorylation at Ser9 results in a kinase-inactive state.³¹ The activity of GSK-3β can also be controlled indirectly through binding to inhibitory complexes with other cytoplasmic proteins,^32,33 or through Wnt-mediated sequestration into multivesicular bodies³⁴ resulting in the physical separation of GSK-3β from its cytoplasmic targets. Control of GSK-3β in the normal state is therefore tightly regulated, with its dysregulation and ensuing aberrant phosphorylation of targets being a common occurrence in many diverse diseases. Several studies have shown that GSK-3β is an important mediator in the injury and repair processes of neurons during cross-talk between DA-neurons and reactive astrocytes.^35,36 These studies showed that astrocyte-derived Wnt1 was capable of blocking GSK-3β activation, allowing the nuclear accumulation of β-catenin and subsequent gene expression of β-catenin-dependent targets essential for neuron survival and repair during chemical or metabolic insults. The importance of regulating the active/inactive states of GSK-3β in regard to neuronal stability is further supported through the analysis of conditional (Tet-inducible) transgenic mice expressing a dominant-negative GSK-3β-K85R mutant or expressing the GSK-3β-S9A mutant.^37,38 In these studies, post-natal Tet-regulated expression of either GSK-3β-K85R or GSK-3β-S9A led to neurodegeneration in the cortex, striatum, and hippocampus. What separates our TG PD model from the tet-inducible GSK-3β models is the spatial patterns of transgene expression, which is influenced by the choice of promoters. The Tet-inducible GSK-3β models are expressed using a CAMKII promoter with our human(h) GSK-3β-S9A transgene being expressed under the Thy-1 promoter. CAMKII-driven expression is limited to neurons originating from the forebrain with Thy-1 promoter-driven expression restricted to neurons in all or most brain regions.^39,40 Although promoter choice effecting tissue expression ultimately decides which regions show degeneration, the important message is that both inactive and hyperactive states of GSK-3β reduce neuronal viability.In our past studies in various in vitro and in vivo models of PD and in postmortem PD tissues, we have consistently observed a positive correlation between increased α-Syn and p-Tau levels with increased GSK-3β-Y216 (the kinase-active form of GSK-3β).^{12, 13, 16, 19, 20} In in vitro studies of MPTP-treated SH-SY5Y cells, blockade of GSK-3β with lithium, or with the highly selective non-ATP competitive inhibitor, TDZD-8, prevented the induction of p-GSK-3β-Y216, abolished p-Tau formation, and reversed the accumulation and aggregation of both p-Tau and α-Syn, averting cell death.¹⁶ Other studies using Rotenone or MPTP/MPP+ in chemical PD models, have shown similar results of decreased neuronal viability during treatments accompanied by dose- and time-dependent increases in GSK-3β activation, with decreased cytotoxicity detected when GSK-3β was inhibited or knocked-down through the use of GSK-3β-specific small molecule inhibitors or through RNAi.^41,42 This suggested to us that p-GSK-3β-Y216 may have a contributory role in the pathogenesis of PD. Using a mouse model overexpressing hGSK-3β-S9A under the Thy-1 promoter together with in vitro kinase assays allowed us to discern the role GSK-3β has in the development of PD-like pathology.⁴³ Analysis of our hGSK-3β-S9A mouse model showed here for the first time that upon aging, these mice develop the cardinal features of parkinsonism, manifested as impaired motor behavior, with associated loss of TH+ neurons, reduced DA production, and shrinkage of SN. Invitro kinase assays confirmed that hGSK-3β was capable of phosphorylating α-Syn on Serine 129 together with the known ability to phosphorylate Tau. Remarkably, both α-Syn and Tau influenced the rate and magnitude of phosphorylation of the other by GSK-3β indicating that an intimate physical relationship exist between the trio of PD related proteins. Together, these data shown indicate the importance of GSK-3β activation, in the behavioral and physiological development of PD like pathology in a new mouse model.     

A Simple Staining Procedure for Detecting the true Acrosome Reaction in Buffalo (Bubalus bubalis) Spermatozoa

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.