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.     

Der Neodarwinismus als axiomatisierbares deduktives System und das Problem der Anpassung und Selektion

In the last decade numerous evolutionary models have been proposed and intensively discussed. Surprisingly, the logical analysis of these different hypotheses is not of great importance in this debate. As a matter of fact, Neodarwinism is a consistent deductive system which is based on few but wellconfirmed axioms. Hence, very often, precise statements and definitions of standpoint can be made. This is exemplified by some repeatedly discussed models concerning the problems of Darwinian »adaptation« and »natural selection« 1. Following the logic of the Neodarwinian theory, extinction can only be the result of overspecialization if some very unusual assumptions are made. 2. The hypothesis of »punctuated equilibrium« GOULD 1982), the »effect hypothesis« (VRBA 1983) and the stochastic evolutionary model SCHOPF 1979) include elements which are logically inconsistent with the »modern synthesis«; they could be »true« only if at least one of the neodarwinian axioms (so far not disproved) is negated.     

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.     

FMRFamide-like immunoreactivity in the brain and pituitary of the teleost. Eigenmannia lineata (Gymnotiformes)

The distribution of cells immunoreactive for the molluscan tetrapeptide FMRFamide in the brain and the pituitary of Eigenmannia was investigated immunohistochemically by the use of the peroxidase-antiperoxidase (PAP) technique and unlabelled antibodies. FMRFi neurons were located in the ganglion of the nervus terminalis at the rostroventral side of the bulbus olfactorius. FMRFi perikarya were also found in a dorsomedial diencephalic nucleus, in the nucleus ventromedialis, in some liquor-contacting neurons of the nucleus lateralis tuberis and of the nucleus recessus lateralis and posterior. The perikarya of the midbrain pre-pacemaker nucleus were only weakly immunoreactive for FMRFamide while large FMRFi neurons (T-cells) occurred in lamina VI of the torus semicircularis, in the brain stem, in dorsal and medial layers of the lobus lineae lateralis posterior (LLLp) and in the medullary electric organ pacemaker nucleus (pm). FMRFi fibers and nerve endings were found in the bulbus olfactorius, in medial areas of the telencephalon, and rather densely in the rostral diencephalon. Ventrocaudally to most of the hypothalamic nuclei the occurrence of immunoreactive fibres increased; many coursed to the pituitary through the pituitary stalk. FMRFi fibres also appeared in the deep layers of the tectum opticum, in the torus semicircularis, in the medial and lateral medulla and below the pacemaker nucleus. Wherever FMRFamide-immunoreactivity occurred fibres and nerve endings could be found in close contact with blood vessels.     

Analysis of biochemical genetic data on Jewish populations: II. Results and interpretations of heterogeneity indices and distance measures with respect to standards.

A nonparametric statistical methodology is used for the analysis of biochemical frequency data observed on a series of nine Jewish and six non-Jewish populations. Two categories of statistics are used: heterogeneity indices and various distance measures with respect to a standard. The latter are more discriminating in exploiting historical, geographical and culturally relevant information. A number of partial orderings and distance relationships among the populations are determined. Our concern in this study is to analyze similarities and differences among the Jewish populations, in terms of the gene frequency distributions for a number of genetic markers. Typical questions discussed are as follows: These Jewish populations differ in certain morphological and anthropometric traits. Are there corresponding differences in biochemical genetic constitution? How can we assess the extent of heterogeneity between and within groupings? Which class of markers (blood typings or protein loci) discriminates better among the separate populations? The results are quite surprising. For example, we found the Ashkenazi, Sephardi and Iraqi Jewish populations to be consistently close in genetic constitution and distant from all the other populations, namely the Yemenite and Cochin Jews, the Arabs, and the non-Jewish German and Russian populations. We found the Polish Jewish community the most heterogeneous among all Jewish populations. The blood loci discriminate better than the protein loci. A number of possible interpretations and hypotheses for these and other results are offered. The method devised for this analysis should prove useful in studying similarities and differences for other groups of populations for which substantial biochemical polymorphic data are available.     

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.