Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.     

The influence of the size and nature of basic activators on Clostridium perfringens polynucleotide phosphorylase-catalysed polyadenylic acid synthesis

1. Basic oligo- and poly-(amino acids) stimulate polyadenylic acid synthesis by purified Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8). 2. The effectiveness of the activators increases with chain length up to approx. 20-30 residues. 3. Polymers of the l and dl series are equally effective on a weight-for-weight basis. 4. l-Lysine, d-lysine, diethylamine and triethylamine, as hydrochlorides or hydrobromides, all stimulate the reaction markedly if their concentration is high enough. Their effect is similar to that of sodium chloride. 5. The size of the product depends primarily on the Mg(2+) concentration and basic polymers have a relatively limited effect on it. 6. Polyadenylic acid itself undergoes an Mg(2+)-catalysed non-enzymic hydrolysis.     

Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization

The bacterial community structure in the winter cover and pelagic zone of a high mountain lake was analyzed by in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes. Cells fixed on membrane filters were hybridized with a probe specific for the domain Bacteria as well as with probes for the alpha, beta, and gamma subclasses of the class Proteobacteria and the Cytophaga-Flavobacterium group. The fraction of bacteria detectable after hybridization with the bacterial probe EUB ranged from 40 to 81% of 4(prm1),6-diamidino-2-phenylindole (DAPI) counts. The bacterial assemblage varied considerably between and within different habitats (snow, slush, and lake water) but was in most cases dominated by members of the beta subclass (6.5 to 116% of bacteria detectable with probe EUB). The sum of bacteria hybridizing with group-specific probes was usually lower than the fraction detectable with probe EUB. Image analysis was used to characterize morphology and the size-specific biomass distribution of bacterial assemblages, which clearly separated the three habitats. Although the measured secondary production parameters and the fraction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride-reducing bacteria varied by more than an order of magnitude in the different slush and pelagic layers, detectability with the fluorescent probe EUB was constantly high. Physiological strategies of bacteria under nutrient limitation and at low temperatures are discussed in the context of the ribosome content of single cells. This study confirms the suitability of fluorescently labeled rRNA-targeted probes for the characterization of bacterial population structures even in oligotrophic habitats.     

Zur Populationsgenetik der sauren Phosphatase der Erythrocyten (E C 3.1.3.2): Phänotypen-und Allelhäufigkeiten in Südwestdeutschland

Summary Within an population sample of 300 individuals of Southwestern Germany the red cell acid phosphatase polymorphism is investigated. Gene frequency estimates are: P^a=0.31, P^b=0.643, P^c=0.047.

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Direktor: Prof. Dr. med. Dr. H. Baitsch

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.     

Active uptake of sodium in the gills of the hyperregulating shore crabCarcinus maenas

Isolated posterior gills of shore crabs,Carcinus maenas, previously acclimated for at least 1 month to brackish water of 10 S, were connected with an artificial hemolymph circulation by means of thin polyethylene tubings. Gills were symmetrically perfused and bathed with 50 % sea water. Transepithelial potential differences (PDs) and fluxes of sodium between medium and blood were measured under control conditions and following reductions of PDs by means of 5 mM internal (blood side) ouabain, 0.5 mM internal and external (bathing medium) NaCN or by exhaustion of energy reserves along with a prolonged perfusion period of more than 9 h. In these experiments²²Na was used as tracer. Each of the three modes of reducing transepithelial potential differences resulted in a decrease in sodium influxes from 500–1000 µmoles g^–1 h^–1 to 250–400 µmoles g^–1 h^–1. The findings suggest that sodium influx, which normally greatly exceeds efflux, was diminished by its active component. The remaining non-inhibitable influx equals efflux values. Our findings thus indicate that efflux is completely passive, while influx has — beside a passive component of efflux magnitudes — an additional active portion which is much larger than the passive component. Since ouabain is a specific inhibitor of the Na-K-ATPase, our results confirm previous findings (Siebers et al., 1985) that the basolaterally located Na-K-ATPase generates the transepithelial potential difference in the gills, which is inside negative by about 6–12 mV. Inhibition of the active portion of sodium influx by internal ouabain along with reduced PDs suggests that transepithelial PDs generated by the branchial sodium pump are the driving force for active sodium uptake in hyperregulating brackish water crabs.     

Domain structure and antiparallel dimers of microtubule-associated protein 2 (MAP2).

We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.     

Aggregation of Aβ40/42 chains in the presence of cyclic neuropeptides investigated by molecular dynamics simulations

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST₁₄) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ₄₂ aggregates. Furthermore, SST₁₄’s presence was also found to promote the formation of toxic Aβ₄₂ oligomers in vitro. In order to elucidate how SST₁₄ influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ₄₂ or Aβ₄₀ peptides with SST₁₄ molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ₄₂ in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ₄₂–SST₁₄, Aβ₄₂–AVP, and Aβ₄₀–SST₁₄. The Aβ₄₂–SST₁₄ mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST₁₄ or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST₁₄ was found to impede aggregation in the Aβ₄₂–SST₁₄ system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ₄₂–SST₁₄ oligomerization.