Solution structures of human and porcine beta-microseminoprotein

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.     

The critically endangered Asiatic cheetah <Emphasis Type="Italic">Acinonyx jubatus venaticus</Emphasis> in Iran: a review of recent distribution,and conservation status

Considerable effort has been put into conservation of the critically endangered Asiatic cheetah Acinonyx jubatus venaticus in Iran during the past few decades, and a thorough review of the species’ status, demography, range and conservation is provided here. We collated a large dataset of all verified occurrence data, photographic records and mortality cases since 1980 throughout the species’ range in Iran. Currently, the cheetah is distributed throughout the arid landscapes of the eastern half of Iran, but the limits of its current and past range as well as population trends are uncertain. Surveys of nearly 40 different areas resulted in 18 localities with confirmed presence of cheetahs in recent years. Camera trapping has been an effective tool to provide evidence of presence and status of cheetahs, revealing the species’ extremely low density and long inter-reserve movements. Together with photographic records, a total of 82 different cheetahs were detected during the 2000s in Iran. Protection status in most areas has been elevated by the Iran government. Asiatic cheetahs are highly vulnerable to extinction, mainly due to causalities mediated by herder persecution, poaching and road collisions as well as prey and habitat loss. Some efforts have been made to address these threats, but range expansion in recent years is a result of greater survey effort, rather than population recovery. We suggest that, despite conservation investment of the last 15 years, the species remains critically endangered on the verge of extinction.     

Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium‐induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH₄⁺ or NO₃^–) or for 3 weeks exposed to exogenous indole‐3‐acetic acid (IAA) or 6‐benzylaminopurine (BAP) at a range of concentrations (10^‐9?10^‐5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant^?1) and specific nodulation (nodules g^?1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4‐fold of the mineral‐N‐free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t‐zeatin (Z) and lower t‐zeatin riboside (ZR) than that the control or nitrate‐treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10^‐7?10^‐5 M strongly inhibited nodulation. However, 10^?9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.     

The ITAM in Nef influences acute pathogenesis of AIDS-inducing simian immunodeficiency viruses SIVsm and SIVagm without altering kinetics or extent of viremia

The role of the immunoreceptor tyrosine-based activation motif (ITAM) that is unique to the Nef protein of the acutely pathogenic simian immunodeficiency virus SIVsmPBj was studied in the context of two AIDS-inducing simian immunodeficiency virus molecular clones. NefY(+) variants of SIVagm9063-2 and SIVsmE543-3 replicated in and induced proliferation of unstimulated pig-tailed macaque PBMC. The pathogenesis of the NefY(+) and NefY(-) clones of SIVagm9063-2, SIVsmE543-3, and PBj6.6 were evaluated by intravenous inoculation of pig-tailed macaques (Macaca nemestrina). Introduction of the ITAM did not increase plasma viral RNA levels nor alter the kinetics of viremia compared with the NefY(-) versions of each clone. Clinical symptoms were not observed in animals inoculated with the NefY(-) variants. In contrast, characteristic PBj symptoms were observed in animals inoculated with any of the three NefY(+) clones. Blunting and fusion of intestinal villi and multifocal infiltration of mononuclear cells were observed in the gastrointestinal tracts of macaques inoculated with the NefY(+) versions. Lesions were associated with active viral replication, as demonstrated by simian immunodeficiency virus-specific in situ hybridization. However, only the macaque inoculated with wild-type NefY(+) SIVsmPBj developed fatal disease; lesions were more widespread and severe in this animal. A switch to macrophages as a viral reservoir and the presence of interleukin-6 in plasma was unique to the macaque infected with PBj6.6. Overall, these data suggest that the ITAM in SIV Nef alters the pathogenesis of simian immunodeficiency virus regardless of the viral background. The change in pathogenesis occurs without enhancement of viral replication. However, NefY(+) variants of SIVagm and SIVsm did not fully recapitulate the virulence of SIVsmPBj, implicating additional viral factors in this unique virus pathogenesis.     

DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.     

Cellular prion protein null mice display normal AMPA receptor mediated long term depression

Cellular prion protein (PrP^C) appears to be involved in numerous physiological processes. We have recently shown a novel modulation of NMDA receptors by PrP^C that results in neuroprotection via silencing of NMDA receptors containing NR2D subunits, whereas no effects on AMPA receptor function could be observed (Khosravani, et al. J Cell Biol 2008; 181:551). Here we show that PrP-null mice show a normal response to long-term depression stimuli requiring AMPA receptor activity, thus further supporting our previous findings of a selective action on NMDA receptors among ionotropic glutamate receptors.Key words: AMPA receptor, NMDA receptor, PrP, long term depression, LTDThe role of prion proteins in the pathophysiology of transmissible spongiform encephalopathies is well documented.¹ Although there is a growing body of literature associating normal cellular prion protein (PrP^C) with functions such as regulation of cell proliferation and survival, cell signalling and immune function,¹ the spectrum of physiological roles attributable to PrP^C remains to be determined. This may in part be due to the fact that mice lacking PrP^C display a relatively mild phenotype, unless subjected to insults such as ischemia or seizures, where increased mortality of the PrP-null mice has been reported.^2–5 Interestingly, the increased neuronal damage in PrP-null mice following excitotoxicity is alleviated upon treatment with the N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) inhibitor MK-801,⁶ suggesting a neuroprotective role of PrP^C via an action on NMDARs, but the mechanism was unclear.We recently described a novel action of PrP^C on NMDAR function.⁷ By examining the neurophysiological properties of hippocampal neurons isolated from PrP-null mice, we were able to show that PrP-null mouse neurons exhibit enhanced and drastically prolonged NMDA evoked currents due to a functional upregulation of NMDARs containing NR2D subunits. Biochemical analyses suggested that NR2D subunits, but not NR2B subunits, co-immunoprecipitated with PrP^C, indicating that PrP^C and NMDARs form physical signaling complexes in neurons. The increased NMDAR function could be phenocopied by RNA interference and were rescued upon overexpression of exogenous PrP^C. The enhanced NMDAR activity resulted in increased neuronal excitability, as well as enhanced glutamatergic-based excitotoxicity in both in vitro and in vivo experiments were neurons were transiently exposed to the selective agonist NMDA. Hence, native PrP^C appears to mediate an important neuroprotective role by virtue of its ability to silence NR2D containing NMDARs. In contrast, minor effects on amplitude and rise and decay-time kinetics were observed for both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and GABA_A (miniature and evoked) currents in synaptically mature hippocampal cultures.AMPA and NMDA receptors have been linked to synaptic plasticity, in particular long term potentiation (LTP) and long term depression (LTD). LTP is believed to mostly reflect a strengthening of the postsynaptic response, caused by a brief period of hyperexcitability that releases significant amounts of glutamate such as during a brief tetanic stimulation. This is thought to result in the opening of AMPA receptors, which depolarize the postsynaptic membrane. This in turn increases the activity of postsynaptic NMDARs, because magnesium ions that normally inhibit NMDAR activity, are dislodged by the postsynaptic depolarization, thus allowing NMDARs to become active. This functional activation of NMDARs results in the influx of calcium ions, which in turn initiate a signaling cascade that results in the membrane insertion of additional AMPA receptors, thus strengthening the synapse. This process is thought to involve NMDA receptor isoforms that predominantly contain the NR2A subunits.⁸A synaptic depotentiation process also can take place that results in the opposite effects of LTP; this process is known as long-term depression or LTD, which also has an NMDAR-dependent component. In contrast to brief tetanic stimulation, as is used in the induction of LTP, establishing LTD requires low frequency stimulation (e.g., 1 Hz for 15 min). Successful and repeatable induction of LTD depends on the parameters used for the conditioning stimulus and more importantly on the age of the animal. In juvenile animals (P12–P21) a low frequency protocol is effective and the mechanism of LTD is believed to depend on the activity of NMDA receptors containing NR2B subunits.⁸ Although a clear distinction of the roles between NR2A and NR2B containing NMDARs, in LTP and LTD respectively, has remained controversial,⁹ it is clear that both NR2A and NR2B are key mediators of alterations in synaptic plasticity. In older animals, the conditioning protocol is reported to require modification to include paired-pulses. This is thought to be due to the involvement of predominantly AMPA (and perhaps kainate) receptors in addition to mGluRs responsible for the synaptic depotentiation (reviewed in ref. ¹⁰).As mentioned earlier, our data obtained from hippocampal cultures indicated only a minor effect of PrP^C knockout on AMPA receptor function. Hence, we hypothesized that AMPA receptor-mediated LTD should be similar in both wild type and PrP-null mice. We therefore examined the effect of PrP on LTD in hippocampal slices obtained from P30–P45 wild type mice and Zurich 1 PrP^C knockout mice. Extracellular potentials were recorded using a patch pipette filled with 150 mM NaCl. First, 10 minutes of baseline evoked (every 30 sec) potentials were recorded to ensure stability of the preparation. LTD was then evoked by application of conditioning paired pulses (Δt = 60 ms) delivered at 1 Hz for 15 min. Thereafter, the field response was sampled every 30 sec for 40 min. As shown in Figure 1, this protocol evoked reliable LTD in wild type mice that partially recovered over the time course of about 20 minutes (Fig. 1). In the PrP^C-null slices, LTD was indistinguishable from that observed in the wild type slices (Fig. 1).Open in a separate windowFigure 1LTD in the CA1 region of hippocampal slices from adult (P30–P45) WT and PrP-null mice. The conditioning pulse (arrow head) was delivered as paired-pulses (Δt = 60 ms) at 1 Hz for 15 min at the Schaffer collaterals. Analysis of field excitatory postsynaptic potential (fEPSP) slope revealed no statistically significant differences (Student''s t-test, p > 0.05) in the extent of induced LTD or the time course of its recovery to baseline. Numbers in parentheses indicate number of slices.The age of the animals, combined with the paired pulse protocol used in our experiments was designed to isolate AMPA receptor mediated LTD.¹⁰ The notion that LTD was unaltered in PrP-null mice is consistent with the observation that AMPA currents were not altered in these mice, and that AMPA receptor-mediated spontaneous synaptic events showed only minute changes compared with wild type animals. These data are also consistent with the notion that PrP-null mice show only mild phenotypes in spatial learning, with no apparent overall short-term memory deficits. Collectively, these data further support a selective action of PrP on NMDA receptors, rather than overall glutamatergic synaptic transmission.