Decreased phosphorylation of a low molecular weight protein by cGMP-dependent protein kinase in variant HL-60 cells resistant to nitric oxide- and cGMP-induced differentiation

We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs [7]. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by ³⁵S-methionine/³⁵S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.     

Late ontogenetic development of adenosine A1 receptor coupling to associated G-proteins in guinea pig cerebellum but not forebrain

The ontogenetic profile of [³H]forskolin and [³H]cyclohexyladenosine ([³H]CHA) binding sites in guinea pig forebrain and cerebellum was investigated. G-protein interactions of these binding sites were also examined by analyzing 5-guanylylimidodiphosphate (Gpp(NH)p) interactions with [³H]CHA and [³H]forskolin binding. In forebrain, similar binding characteristics of [³H]CHA and [³H]forskolin binding are observed between the developmental stages E₃₆ (the earliest time point studied) through to adult (P₂₈, the latest time point studied), although transient increased binding of both ligands is observed just prior to birth. Scatchard analysis of binding isotherms reveal that this transient rise just prior to birth is due to an increase in the number of binding sites (B_max) with little or no change in receptor affinity (K_d) In contrast, in cerebellum both [³H]CHA and [³H]forskolin binding remains at a relatively low level until just prior to birth when a dramatic increase of binding of both ligands is observed which continues to increase up to P₂₈. Scatchard analysis of binding isotherms reveal that such changes in binding of both ligands are largely due to increases in B_max and not K_d, although Scatchard analysis of [³H]CHA binding to cerebellar E₅₁ membranes reveals an absence of higher affinity [³H]CHA binding sites. Gpp(NH)p did not affect [³H]forskolin binding. Gpp (NH)_p displacement profiles of [³H]CHA binding reveal a maximum (adult) inhibition of [³H]CHA binding (approximately 80% displacement) at all time points (E₃₆ through P₂₈) in forebrain membranes, but not in cerebellar membranes. In cerebellum, displacement of [³H]CHA binding by Gpp(NH)p is much greater after birth than before birth. These results suggest that in cerebellum, but not in forebrain, postnatal coupling of adenosine A₁ receptors to associated G-proteins is much more extensive than in the pre-natal period. The extent of this inferred coupling may also coincide with the ontogenetic appearance or presence of [³H]forskolin binding sites.Abbreviations [³H]CHA N⁶-Cyclohexyl-[³H]-Adenosine - Gpp(NH)p–5 Guanylylimidodiphosphate     

Isolation,structure determination and biological activity of A-16686 factors A′ 1, A′ 2 and A′ 3 glycolipodepsipeptide antibiotics

Summary WhenActinoplanes strain ATCC 33076, the producer of A-16686 A1, A2 and A3 complex, is fermented in a suitable medium three additional factors, designated A1, A2 and A3 are produced. These were isolated and characterized, and were shown to differ from the parent components of the original complex by lacking one mannose unit. Bioconversion of A factors into A factors was achieved by incubation with the mycelium ofActinoplanes ATCC 33076. Factor A2 has better antibacterial activity than A2 against some bacteria.     

Restriction analysis of the structural α-L-fucosidase gene and its linkage to fucosidosis

Human alpha-L-fucosidase is a lysosomal enzyme responsible for hydrolysis of alpha-L-fucoside linkages in fucoglycoconjugates. A single gene, FUCA 1, located on chromosome 1p34.1-1p36.1 encodes for alpha-L-fucosidase activity. To gain insight into the nature of the molecular defects leading to fucosidosis, we have characterized the genomic structure of FUCA 1. Restriction-endonuclease analysis suggests that at least seven exons dispersed over 22 kb are present in genomic FUCA 1. Two restriction-fragment-length polymorphisms (RFLPs) have been identified in the Caucasian population. The PvuII and BglI RFLPs each have two codominant alleles in Hardy-Weinberg equilibrium. Allele frequencies for the PvuII RFLP are .70/.30, and those for the BglI RFLP .63/.37. Both RFLPs are in strong linkage disequilibrium with each other, with a correlation coefficient of .94. The polymorphism information content (PIC) of the combined DNA markers is .38, high enough to be useful in the prenatal diagnosis of fucosidosis. The combined lod score for linkage between the fucosidosis mutation and FUCA 1 markers in two families was significant at a recombination fraction of 0. This suggests that the fucosidosis mutation resides in FUCA 1.     

Revision des evolutionsdenkens?

My criticism of the »organism-centered concept of evolution« as exposed once again by Bonik et al. (this vol.) refers to its phylogenetic application but not to the underlying basic assumption. The misunderstanding is due to the fact that Bonik et al. do not distinguish between evolutionary theory (which is concerned with general problems) and phylogenetic research. Nobody would deny that organisms are energy converting systems. Variants with increased energetic efficiency will of course be favoured by natural selection. But this premise alone does not allow to reconstitute phylogenetic lineages. Evolutionary success of a given variant is determined by many other factors as, for instance, the presence or absence of certain predators. Therefore phylogenetic research depends largely on circumstantial evidence from compared anatomy. Finally it should be stressed that the elimination of »abnormal ontogenetic stages« does not contribute to phylogenetic transformation. Bonik et al. are wrong when they believe that this kind of internal selection constitutes a new, hitherto neglected element which necessitates a fundamental revision of evolutionary theory.     

Blood extraction from lancet wounds using vacuum combined with skin stretching.

Key factors and practical limits of blood extraction from lancet wounds on body sites other than the finger were determined by testing a large number of conditions. During these tests, the pain associated with lancing alternate body sites was rated as less painful than a fingerstick 98% of the time. Vacuum combined with skin stretching was effective in extracting an adequate volume of blood from the forearm for glucose testing, up to an average of 16 microl in 30 s. The amount of blood extracted increases with the application of heat or vacuum before lancing, the level of vacuum, the depth of lancing, the time of collection, and the amount of skin stretching. Vacuum and skin stretching led to significant increases, up to fivefold in the perfusion of blood in the skin as measured by laser Doppler. Our observations suggest that vacuum combined with skin stretching increases blood extraction at alternate sites by increasing the lancet wound opening, increasing the blood available for extraction by vasodilatation, and reducing the venous return of blood through capillaries.     

SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism

Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO(2) . The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

Routes to and from the plasma membrane: bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner.