Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution.

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope-NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.

Chloroplast DnaJ-like proteins 3 and 4 (CDJ3/4) from Chlamydomonas reinhardtii contain redox-active Fe-S clusters and interact with stromal HSP70B.

In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the 'green lineage' and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe-S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550-2800 kDa, which appeared to contain RNA. We speculate that the CDJ3-5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.

Myeloid sarcoma in the central nervous system: case report and review of the literature.

Myeloid sarcomas are rare manifestations of mainly myeloblastic leukemia. Their occurrence in the central nervous system is exceptional and current literature is limited to case studies. A case is added herewith and a review was performed to investigate clinical characteristics and treatment options of central nervous system myeloid sarcoma. A 61-year-old female with acute myeloblastic leukemia (FAB M5) and progressive left sided hemiparesis showed a right parieto-occipital epidural lesion mimicking meningioma. Partial resection was performed to reveal a myeloid sarcoma. Reviewing the literature we identified 44 cases with sufficient description of the diagnosis, treatment and follow up to one year. In these patients different treatment regimens were applied. However, when systemic chemotherapy or irradiation was included in the treatment regimen, patients showed the best 1-year survival proportion.

1-Aminocyclopropane-1-carboxylic acid and abscisic acid during the germination of sugar beet (Beta vulgaris L.): a comparative study of fruits and seeds.

The control of sugar beet (Beta vulgaris L.) germination by plant hormones was studied by comparing fruits and seeds. Treatment of sugar beet fruits and seeds with gibberellins, brassinosteroids, auxins, cytokinins, and jasmonates or corresponding hormone biosynthesis inhibitors did not appreciably affect radicle emergence of fruits or seeds. By contrast, treatment with ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) promoted radicle emergence of fruits and seeds. Abscisic acid (ABA) acted as an antagonist of ethylene and inhibited radicle emergence of seeds, but not appreciably of fruits. High endogenous contents of ACC and of ABA were evident in seeds and pericarps of dry mature fruits, but declined early during imbibition. ABA-treatment of seeds and fruits induced seed ACC accumulation while ACC-treatment did not affect the seed ABA content. Transcripts of ACC oxidase (ACO, ethylene-forming enzyme) and ABA 8'-hydroxylase (CYP707A, ABA-degrading enzyme) accumulate in fruits and seeds upon imbibition. ABA and ACC and the pericarp did not affect the seed CYP707A transcript levels. By contrast, seed ACO transcript accumulation was promoted by ABA and by pericarp removal, but not by ACC. Quantification of the endogenous ABA and ACC contents, ABA and ACC leaching, and ethylene evolution, demonstrate that an embryo-mediated active ABA extrusion system is involved in keeping the endogenous seed ABA content low by 'active ABA leaching', while the pericarp restricts ACC leaching during imbibition. Sugar beet radicle emergence appears to be controlled by the pericarp, by ABA and ACC leaching, and by an ABA-ethylene antagonism that affects ACC biosynthesis and ACO gene expression.

The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas.

The vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis of thylakoid membranes in cyanobacteria and plants. VIPP1 and its bacterial ancestor PspA form large homo-oligomeric rings of >1 MDa. We recently demonstrated that VIPP1 interacts with the chloroplast J-domain co-chaperone CDJ2 and its chaperone partner HSP70B, and hypothesized that the chaperones might be involved in the assembly and/or disassembly of VIPP1 oligomers. To test this hypothesis, we analysed the composition of VIPP1/chaperone complexes in Chlamydomonas reinhardtii cell extracts and monitored effects of the chaperones on VIPP1 assembly states in vitro. We found that CGE1, the chloroplast GrpE homologue, is also part of complexes with HSP70B, CDJ2 and VIPP1. We observed that CDJ2-VIPP1 accumulated as low- and high-molecular-weight complexes in ATP-depleted cell extracts, but as intermediate-size complexes in extracts supplemented with ATP. This was consistent with a role for the chaperones in VIPP1 assembly and disassembly. Using purified proteins, we could demonstrate that the chaperones indeed facilitated both the assembly and disassembly of VIPP1 oligomers. Electron microscopy studies revealed that, in contrast to PspA, VIPP1 rings assembled into rod-shaped supercomplexes that were morphologically similar to microtubule-like structures observed earlier in various plastid types. VIPP1 rods, too, were disassembled by the chaperones, and chaperone-mediated rod disassembly also occurred when VIPP1 lacked an approximately 30-aa C-terminal extension present in VIPP1 homologues but absent in PspA. By regulating the assembly state of VIPP1, the chloroplast HSP70 chaperone system may play an important role in the maintenance/biogenesis of thylakoid membranes.

Trigeminal neuralgia in two patients with glioblastoma.

Headache in glioblastoma patients often indicates raised intracranial pressure by either tumor edema or tumor progression. We report local glioblastoma growth causing cranial nerve lesions as well as trigeminal neuralgia, and highlight pain management in these patients.

Unilateral compression neuropathy of the hypoglossal nerve due to head suspension orthosis in mitochondriopathy.

An 85-year-old woman with multisystem mitochondriopathy experienced tension headache, cervical pain, torque head-posture, and vertigo since 1980 for which she was continuously wearing a head-suspension-orthosis- since 1990. Since 1996 she developed severe left-sided weakness and wasting of the tongue. Needle-EMG of the left genioglossus muscle revealed abnormal spontaneous activity and reduced interference-pattern. No morphological alterations in the anatomical course of the hypoglossal nerve were found. Severe, unilateral weakness and wasting of the tongue was interpreted due to chronic compression of the hypoglossal nerve by long-standing use of a head-suspension-orthosis for cervical pain from cervical muscle weakness and resulting spinal degeneration.

Transmyocardial laser revascularization and left ventricular reduction surgery affect ventricular arrhythmias and heart rate variability.

BACKGROUND: Transmyocardial laser revascularization (TMLR) and left ventricular reduction by endoventricular patch plasty (LVR) are 2 new surgical procedures performed in patients with endstage coronary artery disease and left ventricular dilation/aneurysms, respectively. As these are performed in patients at high risk for sudden cardiac death and may interact with arrhythmogenesis, we assessed the influence of these procedures on incidence and severity of ventricular tachyarrhythmias and time-domain heart rate variability. METHODS: Preoperative and one week postoperative 24-hour Holter recordings were performed in 37 patients undergoing TMLR (n = 23, CO2-laser technique) or LVR (n = 14). RESULTS: TMLR patients received a mean of 27.2 +/- 9.2 laser channels. Postoperatively, the proportion of patients who underwent TMLR with spontaneous ventricular tachycardia (> or =4 repetitive ventricular beats) increased (0% vs 26%, P <.05), including one patient who died from documented ventricular fibrillation during monitoring. There was no correlation to the number and/or location of laser-induced channels or to perioperative CK levels. HRV parameters were not altered by TMLR. By contrast, LVR did not significantly influence ventricular tachyarrhythmia episodes but markedly depressed all major HRV parameters (SDNN 116.4 vs 61.8, RMSSD 35.2 vs 19.9, pNN50 14.5 vs 4.9, all P <.05). CONCLUSIONS: Early after TMLR, there is evidence of an increased incidence of spontaneous ventricular tachycardia enhancing the risk for sudden cardiac death, while HRV remains unaffected. By contrast, LVR resulted in a marked reduction in HRV still present one week postoperatively, while no effect was observed on incidence and/or severity of spontaneous ventricular tachyarrhythmias.