The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Alpha-melanotropin in the frog brain: regional distribution, quantification and subcellular distribution

The distribution of alpha-MSH containing neurons was studied by immunofluorescence in the brain of the frog Rana ridibunda. Most immunoreactive cell bodies were found in the ventral hypothalamic area. A rich network of fluorescent fibers was observed in the ventral infundibular region, coursing towards the preoptic area and the ventral telencephalon. Some fibers, directed backwards, project into median eminence. By means of a specific radioimmunoassay, the concentrations of alpha-MSH immunoreactive material has been determined in 10 different regions of the brain. The highest concentrations were observed in the infundibular and the preoptic regions. Using the immunogold technique, electron microscopy showed that immunostaining was restricted to 70-100 nm dense core vesicles in positive cell bodies and fibers. These results suggest that, in addition to well known hormonal (melanotropic) activity, alpha-MSH could play the role of a neurotransmitter in the frog brain.     

Pregnancy incidence and correlates during the HVTN 503 Phambili HIV vaccine trial conducted among South African women

Background

HIV prevention trials are increasingly being conducted in sub-Saharan Africa. Women at risk for HIV are also at risk of pregnancy. To maximize safety, women agree to avoid pregnancy during trials, yet pregnancies occur. Using data from the HVTN 503/“Phambili” vaccine trial, we report pregnancy incidence during and after the vaccination period and identify factors, measured at screening, associated with incident pregnancy.

Methods

To enrol in the trial, women agreed and were supported to avoid pregnancy until 1 month after their third and final vaccination (“vaccination period”), corresponding to the first 7 months of follow-up. Unsterilized women, pooled across study arms, were analyzed. Poisson regression compared pregnancy rates during and after the vaccination period. Cox proportional hazards regression identified associations with first pregnancy.

Results

Among 352 women (median age 23 yrs; median follow-up 1.5 yrs), pregnancy incidence was 9.6/100 women-years overall and 6.8/100 w-yrs and 11.3/100 w-yrs during and after the vaccination period, respectively [Rate Ratio = 0.60 (0.32–1.14), p = 0.10]. In multivariable analysis, pregnancy was reduced among women who: enrolled at sites providing contraception on-site [HR = 0.43, 95% CI (0.22–0.86)]; entered the trial as injectable contraceptive users [HR = 0.37 (0.21–0.67)] or as consistent condom users (trend) [HR = 0.54 (0.28–1.04)]. Compared with women with a single partner of HIV-unknown status, pregnancy rates were increased among women with: a single partner whose status was HIV-negative [HR = 2.34(1.16–4.73)] and; 2 partners both of HIV-unknown status [HR = 4.42(1.59–12.29)]. Women with 2 more of these risk factors: marijuana use, heavy drinking, or use of either during sex, had increased pregnancy incidence [HR = 2.66 (1.24–5.72)].

Conclusions

It is possible to screen South African women for pregnancy risk at trial entry. Providing injectable contraception for free on-site and supporting consistent condom use may reduce incident pregnancy. Screening should determine the substance use, partnering, and HIV status of both members of the couple for both pregnancy and HIV prevention.

Trial Registration

SA National Health Research Database DOH-27-0207-1539; Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00413725","term_id":"NCT00413725"}}NCT00413725     

Diminished bronchial reactivity to cold air in diabetic patients with autonomic neuropathy.

To investigate the role of neural pathways in the nonasthmatic response to eucapnic hyperventilation with below freezing air five diabetic patients with severe symptomatic autonomic neuropathy were studied. Their responses were compared with those shown by five diabetic patients without autonomic neuropathy and five non-diabetic controls. After bronchial provocation testing with cold air the diabetic patients with autonomic neuropathy did not show a significant fall in specific airways conductance (mean (SE) maximum percentage fall 2.0 (3)%), whereas conductance fell in the diabetic patients without neuropathy by 30.8 (2.0)% (p less than 0.001) and in the non-diabetic controls by 22.7 (4.6)% (p less than 0.02). In subjects who do not have asthma the bronchial response to cold air is mediated largely via neural mechanisms.     

Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion

We investigated the mechanisms by which two nitric oxide (NO) donors, diethylenetriamine/NO adduct (DETA/NO) and S-nitrosoglutathione (GSNO), induced cell death in a J774 macrophage cell line. Both NO donors induced caspase activation within 6 h, but only DETA/NO-induced caspase activation was sensitive to inhibition of p38 and was completely prevented by antioxidants catalase, ascorbate, dehydroascorbate, or N-acetylcysteine, suggesting that DETA/NO-induced apoptosis may be mediated by H(2)O(2). Consistent with this, DETA/NO acutely stimulated reactive oxygen species (ROS) production by mitochondria and cells, and inhibited catalase-mediated H(2)O(2) breakdown in cells. After prolonged, 24 h exposure of cells to DETA/NO, inactivation of caspases occurred, which was accompanied by an increase in necrosis. DETA/NO-induced necrosis was insensitive to caspase inhibitors, but was partially prevented by catalase or N-acetylcysteine, and was preceded by inhibition of glyceraldehyde-3-phosphate dehydrogenase and a decrease in cellular adenosine triphosphate (ATP). GSNO was even more potent in inhibiting glycolysis and switching apoptosis to necrosis. In cells depleted of glutathione, GSNO and DETA/NO induced rapid necrosis, which resulted from rapid depletion of ATP due to inhibition of glycolysis. Glycolytic intermediate 3-phosphoglycerate decreased DETA/NO-induced necrosis and increased apoptosis. We conclude that: (i). NO-induced apoptosis is mediated by H(2)O(2); (ii). NO-induced necrosis is mediated by energy failure speeded by thiol depletion.