Comparison of the solution and crystal structures of mitochondrial cytochrome c. Analysis of paramagnetic shifts in the nuclear magnetic resonance spectrum of ferricytochrome c

The two accompanying papers describe the assignment of methyl-containing spin-systems in the 1H nuclear magnetic resonance spectra of tuna ferricytochrome c and tuna ferrocytochrome c. At present, 104 resonances from 208 C-H protons are assigned in both oxidation states. In this paper, the difference in chemical shift of a resonance between the two oxidation states is used together with a dipolar model of the unpaired electron spin of ferricytochrome c to compare the structure of cytochrome c in solution with three high-resolution structures of cytochrome c obtained by X-ray diffraction in single crystals. The overall protein fold and the positions of most of the haem-packing residues are shown to be invariant between the crystal and solution. However, three regions of the protein, at the C terminus, around the haem propionic acid groups and at the haem crevice near thioether-2, are found to undergo conformational changes on the removal of crystal packing constraints.     

A determinant of polyomavirus virulence enhances virus growth in cells of renal origin.

We have identified a strain of polyomavirus, Py(L), which is unusual in causing acute morbidity and early death after inoculation of newborn mice. We determined that these animals died of kidney failure associated with extensive, virus-mediated destruction of renal tissue. Interestingly, the Py(L) strain infects baby mouse kidney cell cultures more efficiently than do other strains.     

Applications of fluorochromes to pollen biology. II. The DNA probes ethidium bromide and Hoechst 33258 in conjunction with the callose-specific aniline blue fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.     

14C labelling of octulose bisphosphates by L-type pentose pathway reactions in liver in situ and in vitro

The complete reaction sequence of the pentose pathway in vitro was studied by incubating [1-14C] ribose 5-phosphate with rat liver enzyme preparation and assessed by both the rate and extent of formation of the glucose 6-P product. The reactions formed, as intermediates, the 1,8-bisphosphates of D-glycero D-ido octulose (D-g D-i Oct) and D-glycero D-altro octulose, both heavily labelled at C-4 with 14C isotope during the 12h incubation. The formation of the octulose phosphates and the specificity of their isotopic labelling confirms an important prediction of, and contribution by reactions of the L-type pentose phosphate pathway (L-PP) in liver in vitro. Infusion in situ of [6-14C] glucose into the liver of the anaesthetized rabbit resulted in the formation of high specific activity [8-14C] D-g D-i Oct 1,8-P2. The specificity of labelling indicates that the octulose intermediate is formed according to the options of the L-PP mechanism of glucose metabolism in intact liver.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Evidence from cDNA clones that the rat leukocyte-common antigen (T200) spans the lipid bilayer and contains a cytoplasmic domain of 80,000 Mr

The leukocyte-common antigen (L-CA or T200) includes a family of lymphoid and myeloid cell surface glycoproteins with apparent molecular weights from 180,000 to 240,000. We report a partial protein sequence for thymocyte L-CA containing 1073 amino acids predicted from cDNA clones isolated using an oligonucleotide probe. Only one segment (residues 347-368) is likely to cross the membrane, and peptide data suggest that sequences N-terminal to this are outside the cell, with residues 369-1073 inside. The cytoplasmic domain includes possible phosphorylation sites and an internal homology between residues 385-671 and 676-986. Analysis of B lymphocyte cDNA clones suggests that B cell and thymocyte mRNAs are identical in 3' sequences, but size differences in Northern blots suggest 5' sequences may differ.     

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.     

Butyrate formation from glucose by the rumen protozoon Dasytricha ruminantium.

Production of butyrate by the holotrich protozoon Dasytricha ruminantium involves the enzymes of glycolysis, pyruvate:ferredoxin oxidoreductase, acetyl-CoA:acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxyacyl-CoA hydro-lyase, 3-hydroxyacyl-CoA reductase, phosphate butyryltransferase and butyrate kinase. Subcellular fractionation by differential and density-gradient centrifugation on sucrose gradients indicated that all those enzymes except pyruvate:ferredoxin oxidoreductase were non-sedimentable at 6 X 10(6) g-min. Butyrate kinase and phosphate butyryltransferase were associated with the large- and small-granule fractions. Thus, although metabolic reactions necessary for butyrate production proceed predominantly in the cytosol, hydrogenosomes play a key role in the conversion of pyruvate into acetyl-CoA.     

Male-to-male transmission of the velo-cardio-facial syndrome: a case report and review of 60 cases

The velo-cardio-facial syndrome is one of the most common syndromes of clefting. Previous reports have shown vertical pedigree transmission, but in all cases the gene was maternally transmitted. The genetics of this syndrome had been suspected as autosomal dominant, but X-linked dominant inheritance could not be ruled out. This report describes an instance of male-to-male transmission of the velo-cardio-facial syndrome. In addition, the clinical findings in 60 cases are reported to further delineate the phenotypic spectrum of the syndrome.