Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

The conservation of poly-A-containing RNA during the dormant state of the moss Polytrichum commune.

The moss Polytrichum commune can be dried to less than 2% of free water and kept for some weeks without losing its viability. Upon rehydration of the moss, protein synthesis starts about 60 minutes before incorporation of radioactive precursors into RNA can be observed. Pulse labeled total RNA and poly-A-containing RNA do not show quantitative or qualitative alterations during desiccation up to 20 days.     

Citrulline metabolism in normal and citrullinemic human lymphocyte lines

Citrullinemia is one of the five aminoacidurias associated with the Krebs-Henseleit urea cycle. A long-term lymphocyte line (UM-21) derived from a patient with this disease and nine of ten clones of this line were found to have no activity for the enzyme argininosuccinate synthetase (AS), as demonstrated by their inability to grow in medium in which citrulline had been substituted for arginine, by their inability to incorporate arginine-C14 derived from citrulline-C14 into cellular protein, and by direct enzyme assay. One clone had normal or nearly normal argininosuccinate synthetase activity, as demonstrated by the same criteria. Nutritional "variants" able to grow logarithmically in medium containing citrulline were isolated from UM-21 and three clones. The apparent Kms of AS for citrulline in UM-21, the ten clones, the variant lines, and a normal line were measured and fell into three groups: AS in UM-21 and nine clones had no measurable apparent Km for citrulline; AS in the variant cells had apparent Kms for citrulline of approximately 20 mM; and AS in the normal cell line and one clone had apparent Kms for citrulline of 0.2 mM. The data suggest that the defect in the citrullinemic cell lines is due to a mutation in the structural gene coding for argininosuccinate synthetase.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

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Peptide Mimetic of the S100A4 Protein Modulates Peripheral Nerve Regeneration and Attenuates the Progression of Neuropathy in Myelin Protein P0 Null Mice

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P₀ null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.     

Synthesis and Application of Negatively Charged PNA Analogues

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.