Ribosome specificity of archaebacterial elongation factor 2. Studies with hybrid polyphenylalanine synthesis systems

Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus. By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom. In contrast, EF-1 from T. thermophilus significantly cooperated with ribosomes from T. acidophilum.     

The neurosecretory system of the adult Calliphora erythrocephala

Summary Neurosecretory cells in the brain and suboesophageal ganglion of the adult female Calliphora erythrocephala have been studied in the light microscope. The paraldehydefuchsin stain (PAF) gave by far the clearest pictures.The medial neurosecretory cells of the protocerebrum (m.n.c.) show definite cyclic changes as to the size of the nuclei and the content of secretory material. The cytological changes depend on the age of the fly and the diet given and are correlated with ovarian development.The nuclear size is held to express the metabolic activity of the cells. Cells with large nuclei, as found in young sugar-flies (S1D) and meat-fed flies with developing ovaries (S4D/M2D), contain less secretory material which is released through the axons, while the m.n.c. of old sugar-flies (S6D) have small nuclei and are stuffed with secretory material which is stored in the perikarya.These results confirm those obtained by darkfield microscopy of living m.n.c. by Lea and E.Thomsen (1962 and unpublished).No really convincing evidence for the existence of more than one type of m.n.c. was found.Two small groups of lateral cells were observed. Possibly neurosecretory are further: 1-(2) cells at the base of each optic lobe, two groups of 2–3 cells on the caudal side of the brain, and 2 cells ventrally in the suboesophageal ganglion.Giant neurons of unknown function are situated very near the m.n.c. Their axons join those from the m.n.c., but end in the suboesophageal ganglion.The same region comprises a number of peculiar cells, each containing a large, fluid-filled vacuole (the vacuolated cells). Similar cells are associated with the possibly neurosecretory cells on the caudal side of the brain.My sincere thanks are due to my wife Dr. Ellen Thomsen, who made all the excisions of the brains and the measurements of the nuclei, to T.C.Normann for valuable assistance with the photographic work, and to Mrs. K. Bahnert for technical help with the gallocyanin method. The Carlsberg Foundation has supported the work with grants.     

Evolution of the HLA-DR1 gene family. Structural and functional analysis of the new allele "DR-BON".

The molecules of the MHC are highly polymorphic and involve in Ag presentation; their striking genetic polymorphism allows probable interactions with a large variety of antigenic fragments when these are presented to the TCR. It is therefore of interest to explore the extent of this polymorphism and the mechanisms of its generation. We have studied the class II HLA-DR blank allele DR-BON that has been previously defined by MLR, restriction fragment length polymorphism, and two-dimensional gel electrophoresis. A cDNA library was constructed from a DR-BON homozygous typing cell and cDNA corresponding to DR alpha- and DR beta-chains were sequenced. By comparison with other known alpha- and beta-chain sequences it is shown that the alpha-chain is invariant and the beta-chain differs from DR1 by only six nucleotides, clustered in the third variable region with three amino acid changes at position 67, 70, and 71. The short DNA stretch of sequence encoding the 67-71 region is also present in other DR alleles: DR4/Dw10, DRw13, and some DRw11 specificities. Therefore we propose that a gene conversion-like event has occurred between the DRB1 *0101 (DR1/Dw1) gene and one of these three DRB1 genes. Extensive typing has been performed with a DR-BON-specific 17-mer oligonucleotide. Cross-hybridization with other genes than the ones expected from DNA sequence comparison was not observed. A selected panel of DR-BON reactive T cell clones shows three patterns of reactivity. Some clones are strictly DR-BON specific; some cross-reacted with DRw13 and a few cross-reacted with other haplotypes. The role of different epitopes of the third variable region of HLA-DR beta chain in allo-reaction is discussed.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Isolation of region-specific probes from pig Chromosome 6 by coincidence cloning

Coincidence cloning is a technique that permits the isolation of sequences common to two independent sources of complex DNA, and this method has been used to isolate a set of probes from a region of porcine Chromosome (Chr) 6 containing the loci for glucosephosphate isomerase (GPI) and the skeletal muscle calcium release channel (CRC). Porcine DNA was specifically PCR-amplified from a pigxhamster hybrid cell line containing the centromere region (p1.2–q1.2) of pig Chr 6 and other pig chromosome fragments by use of a porcine SINE specific primer with an EcoRI site in the 5-end. Flow-sorted Chr 6 preparations were amplified with the same SINE primer, but with a SalI site in the 5-end. The products were digested with EcoRI and SalI respectively, combined, denatured, and reannealed. The heteroduplex molecules, containing both an EcoRI and a SalI cohensive end, were selected by cloning in SalI/EcoRI-digested pUC13. Approximately 40% of the primary clones contained a single SalI/EcoRI-insert, indicating that they are coincidence clones. The average insert size was 1.4 kb. Fluorescence in situ hybridization of a pool of 34 coincidence clones to pig chromosomes showed a preferential labeling of the centromere region and of the q2.5–q2.7 region of pig Chr 6. Nineteen coincidence clones were hybridized to SINE-PCR products from flow-sorted pig Chr 6 and to pigxrodent hybrid cell lines. Eighteen clones gave positive signals correlated with the GPI/CRC content of the source DNAs.     

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A₁ adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a L_i value for A₁ receptor binding of <1 nM.     

Assignment of the dipeptidylpeptidase IV (DPP4) gene to pig Chromosome 15q21

A porcine 2-kb partial dipeptidylpeptidase IV (DPP4, EC 3.4.14.5) cDNA clone and a porcine 16-kb genomic fragment containing parts of the DPP4 gene were isolated, characterized, and used as probes to map the DPP4 gene to pig Chr (Chr) 15q21 by fluorescence in situ hybridization. A two-allele RFLP was revealed for the DPP4 gene. This polymorphism was utilized in a linkage test against the erythrocyte antigen G (EAG), previously assigned to Chr 15, and the microsatellite S0088, which is linked to EAG. The linkage analyses revealed significant evidence for linkage confirming the assignment of DPP4 to Chr 15.