The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Comparative genome analysis of four magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function

Magnetotactic bacteria (MTB) are a heterogeneous group of aquatic prokaryotes with a unique intracellular organelle, the magnetosome, which orients the cell along magnetic field lines. Magnetotaxis is a complex phenotype, which depends on the coordinate synthesis of magnetosomes and the ability to swim and orient along the direction caused by the interaction with the Earth's magnetic field. Although a number of putative magnetotaxis genes were recently identified within a conserved genomic magnetosome island (MAI) of several MTB, their functions have remained mostly unknown, and it was speculated that additional genes located outside the MAI might be involved in magnetosome formation and magnetotaxis. In order to identify genes specifically associated with the magnetotactic phenotype, we conducted comparisons between four sequenced magnetotactic Alphaproteobacteria including the nearly complete genome of Magnetospirillum gryphiswaldense strain MSR-1, the complete genome of Magnetospirillum magneticum strain AMB-1, the complete genome of the magnetic coccus MC-1, and the comparative-ready preliminary genome assembly of Magnetospirillum magnetotacticum strain MS-1 against an in-house database comprising 426 complete bacterial and archaeal genome sequences. A magnetobacterial core genome of about 891 genes was found shared by all four MTB. In addition to a set of approximately 152 genus-specific genes shared by the three Magnetospirillum strains, we identified 28 genes as group specific, i.e., which occur in all four analyzed MTB but exhibit no (MTB-specific genes) or only remote (MTB-related genes) similarity to any genes from nonmagnetotactic organisms and which besides various novel genes include nearly all mam and mms genes previously shown to control magnetosome formation. The MTB-specific and MTB-related genes to a large extent display synteny, partially encode previously unrecognized magnetosome membrane proteins, and are either located within (18 genes) or outside (10 genes) the MAI of M. gryphiswaldense. These genes, which represent less than 1% of the 4,268 open reading frames of the MSR-1 genome, as yet are mostly of unknown functions but are likely to be specifically involved in magnetotaxis and, thus, represent prime targets for future experimental analysis.     

Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR-gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity.     

Development of non-phosphorylated cyclic thioether peptide binding to the Grb2-SH2 domain

Summary One of the critical intracellular signaling pathways involves specific interactions between growth factor receptors and the adaptor protein Grb2. These interactions normally involve specific tyrosine phosphorylated regions in receptors and other cognate proteins. Following the lead of our recent findings that a phage library based non-phosphorylated disulfide linked 11-mer peptide inhibited such interactions, we report here the synthesis of novel redox-stable cyclic peptide analogs. These include thioether cyclized and backbone cyclized structures. The thioether analog was prepared under mild conditions from an N-terminally chloroacetylated and C-terminally cysteine extended peptide precursor. The thioether peptide showed equipotent binding affinity for the Grb2-SH2 domain (IC₅₀=10–15 μM) when compared to the disulfide cyclized lead-peptide. The bioactive thioether linked peptide was demonstrated to offer advantages to the disulfide cyclized peptides under physiological conditions.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

DOPA, dopamine, and DOPAC concentrations in the rat gastrointestinal tract decrease during fasting

The aim of the present study was to test the hypothesis that 3, 4-dihydroxyphenylalanine (DOPA) and dopamine (DA) in the gastrointestinal tract are to a large extent of exogenous origin and derived from food. Tissue concentrations of norepinephrine (NE), epinephrine (Epi), DA, DOPA, and 3,4-dihydroxyphenylacetic acid (DOPAC), as measured by reverse-phase HPLC with electrochemical detection, were studied in fed and 4-day-fasted Wistar rats as well as in sympathectomized and adrenodemedullated rats. Sympathectomy and adrenal demedullectomy decreased tissue concentrations of NE and Epi, respectively, but had no effect on the level of tissue DOPA. Large amounts of DOPA and DA were present in the gastrointestinal tract. Fasting decreased DOPA and DA in the stomach and DOPA concentrations in the quadriceps muscle but no concentrations in other organs. DOPAC in the heart decreased both in response to sympathectomy and to fasting, whereas DOPAC decreased in plasma after fasting and in skeletal muscle after sympathectomy. We conclude that the food content of DOPA and DA is of major importance for the metabolism of DA and, thus, for the dopamine-sulfate content in the gastrointestinal tract and in plasma. The decrease in muscle DOPA after fasting may be explained by less insulin being available during fasting for stimulation of DOPA uptake in the muscle depot. DOPAC in the organism seems to be of a dual origin, derived partly from DA in the food and partly from DA synthesized in sympathetic nerves.     

Biphasic effect of protein kinase C activators on spontaneous and glucocorticoid-induced apoptosis in primary mouse thymocytes

Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.