Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules

Chartins are a unique class of three families of microtubule-associated proteins, each consisting of several isoforms possessing varying degrees of phosphorylation. The most highly phosphorylated chartin isoforms are highly enriched in neuronal cell fractions containing microtubules and there is evidence that their phosphorylation may play a role in promoting neurite outgrowth. The present work describes the relationship between the phosphorylation state of chartins and the presence of intact microtubules in long-term cultures of NGF-treated, neurite-bearing PC12 cells. Cultures were depleted of microtubules by exposure to high concentrations of depolymerizing agents for 2-24 h. Radiolabeling of cellular proteins with [32P]orthophosphate or [35S]methionine revealed that both the ongoing and steady-state phosphorylation of chartins is markedly altered under these conditions. Two-dimensional isoelectric focusing by SDS-PAGE of whole cell extracts demonstrated that the more acidic, highly phosphorylated isoforms are diminished with a concomitant increase in the more basic, less phosphorylated isoforms. These phosphorylation changes were relatively specific for the chartins and were not observed for phosphorylated MAP 1.2, phospho-beta-tubulin, or most other phosphoproteins. Thus, the phosphorylation state of chartins, but not of other phosphoproteins, is regulated by the presence of native microtubules. Despite depolymerization of microtubules, neurites remained extended for at least 24 h. Neurite elongation, however, was arrested. Microtubules, therefore, may be required for extension, but not for short-term maintenance of well-established neurites. Taxol, which promotes tubule assembly and stability, does not, conversely, drive phosphorylation of the chartins. Instead, taxol appeared to decrease the turnover of phosphate in microtubule-associated, acidic chartin isoforms. These data suggest several models as to how chartin phosphorylation is regulated in neurite-bearing cells and indicate that phosphorylation of cytoplasmic and microtubule-associated chartins occurs via different mechanisms.     

Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein. Regulation by nerve growth factor and other agents

Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.     

Orientation responses of the grasshopper, Melanoplus sanguinipes, to visual, olfactory and wind stimuli and their combinations

Prereproductive adults of the grasshopper, Melanoplus sanguinipes (F.) (Orthoptera, Acrididae), demonstrated orientation and movement towards both visual and olfactory stimulus sources in a still-air chamber. Visual stimuli (wheat and lima bean foliage, vertical black or yellow-green stripes, and a yellow-green broad leaf pattern) were approached more frequently than the control white background surface. Olfactory stimuli (chopped wheat foliage and a four-component, synthetic, grass odor blend of volatiles) elicited an even greater positive response than the visual stimuli. Changing the proportions of the four volatiles in the blend significantly reduced positive orientation responses to the stimulus source. Visual cues of wheat foliage and olfactory cues of either chopped wheat odor or the grass odor blend gave greater responses when combined than when presented separately.In flowing air or wind, nearly all insects demonstrated a rapid positive response to odors of chopped wheat and the grass odor blend, significantly greater than the response to the same stimuli in still air. However, positive responses to visual cues were not significantly greater in wind than in still air. When combined with the olfactory stimuli in flowing air, visual cues did not increase the incidence of response. Grasshoppers responding to grass odors in wind moved more rapidly and directly toward the source, and stopped less often and for shorter durations than insects responding to odor in still air or to visual cues.We conclude from these studies that M. sanguinipes adults show orientation behavior to both visual and olfactory stimuli from food plant sources, although leaf odors elicit a stronger positive response particularly when carried by wind.     

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.     

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.     

Hepatitis E virus as a Cause of Acute Hepatitis in The Netherlands

Background

Recent studies indicate that 27% of Dutch blood donors have evidence of past infection with HEV. However, the low number of diagnosed HEV infections indicates either an asymptomatic course or under diagnosis.

Objectives

We investigated whether HEV is a cause of acute hepatitis in Dutch patients and which diagnostic modality (serology or PCR) should be used for optimal detection.

Study design

Serum samples were retrospectively selected from non-severely immuno-compromised patients from a university hospital population, suspected of having an infectious hepatitis. Criteria were: elevated alanine aminotransferase (ALT> 34 U/l) and request for antibody testing for CMV, EBV or Hepatitis A (HAV).

Results

All samples were tested for HEV using ELISA and PCR. Ninety patients/sera were tested, of which 22% were HEV IgG positive. Only one serum was IgM positive. HEV PCR was positive in two patients: one patient was both HEV IgM and IgG positive, the other patient was only IgG positive. Both HEV RNA positive samples belonged to genotype 3. Evidence of recent infection with CMV, EBV and HAV was found in 13%, 10% and 3% respectively.

Conclusions

Although our study is limited by small numbers, we conclude that HEV is a cause of acute hepatitis in hospital associated patients in The Netherlands. Moreover, in our study population the prevalence of acute HAV (3%) was almost similar to acute HEV (2%). We propose to incorporate HEV testing in panels for acute infectious hepatitis. Negative results obtained for HEV IgM in a HEV PCR positive patient, indicates that antibody testing alone may not be sufficient and argues for PCR as a primary diagnostic tool in hospital associated patients. The high percentage of HEV IgG seropositivity confirms earlier epidemiological studies.     

Strong genetic isolation despite wide distribution in a commercially exploited coastal shark

The common smoothhound, Mustelus mustelus, is an epibenthic species targeted by fisheries around the world driven by the increasing demand for shark products. Given the wide-spread occurrence of this species and corresponding lack of molecular data in many areas of said distribution, baseline molecular assessments of this commercially important shark may contribute to finer-scale analyses in areas in which this species is targeted. Therefore, population genetic analyses were conducted along the East Atlantic, from the Mediterranean Sea to the south-east coast of Africa, using microsatellite markers and the mitochondrial control region (mtCR). Overall, M. mustelus displayed low to moderate genetic diversity, with the Mediterranean populations appearing to exhibit the lowest mitochondrial diversity, and the west African populations displaying the lowest nuclear diversity. Microsatellite analysis indicated strong genetic differentiation between the three regions, with finer-scale population structure in each region, without correlation between genetic and geographical distance. For the mtCR sequences, a total of 18 haplotypes were identified, with a high degree of divergence discernable between the regions, largely in accordance with the microsatellite data. The study documents a remarkable level of population isolation across a vast area, suggesting little or no present-day connectivity among extant populations. The findings may serve as an essential baseline for global population management and commercial traceability of this threatened shark.

     

Structure of the species--energy relationship

The relationship between energy availability and species richness (the species-energy relationship) is one of the best documented macroecological phenomena. However, the structure of species distribution along the gradient, the proximate driver of the relationship, is poorly known. Here, using data on the distribution of birds in southern Africa, for which species richness increases linearly with energy availability, we provide an explicit determination of this structure. We show that most species exhibit increasing occupancy towards more productive regions (occurring in more grid cells within a productivity class). However, average reporting rates per species within occupied grid cells, a correlate of local density, do not show a similar increase. The mean range of used energy levels and the mean geographical range size of species in southern Africa decreases along the energy gradient, as most species are present at high productivity levels but only some can extend their ranges towards lower levels. Species turnover among grid cells consequently decreases towards high energy levels. In summary, these patterns support the hypothesis that higher productivity leads to more species by increasing the probability of occurrence of resources that enable the persistence of viable populations, without necessarily affecting local population densities.