Molecular organization and haplotype analysis of centromeric DNA from human chromosome 17: Implications for linkage in neurofibromatosis

The alpha satellite DNA subset located at the centromere of human chromosome 17 has been shown to be tightly linked genetically to the gene for von Recklinghausen neurofibromatosis (NF1). The centromeric DNA polymorphisms used for linkage analyses in NF1 are complex and involve a "locus" (D17Z1) that spans over one million base pairs of satellite DNA. To understand more completely the basis for these polymorphisms and how they might be best scored and used in the analysis of NF1, we have examined the molecular composition of the alpha satellite array on individual copies of chromosome 17 by two complementary approaches. First, we have analyzed segregation of chromosome 17 alpha satellite haplotypes in large, three-generation families that provide information on the different types of alpha satellite segregating in a block fashion. Second, we have analyzed directly the extent of variation in different D17Z1 arrays by genomic blotting analysis of haploid copies of chromosome 17 isolated in rodent/human somatic cell hybrids. The data indicate the existence of a wide range of different alpha satellite variants on individual copies of chromosome 17, each haplotype differing in the size, restriction map, and relative proportion of particular polymorphic repeat forms. Despite this complexity, the D17Z1 markers provide a potentially useful and genetically close starting point for the molecular and clinical analysis of NF1.     

Frequencies of IL-2- and IL-4-secreting T cells in naive and antigen-stimulated lymphocyte populations

The relative frequencies of IL-2-and IL-4-secreting precursors in naive and Ag-primed populations were investigated by using limiting dilution analysis. Cells capable of IL-4 production in lymphoid populations freshly isolated from mice were rare in comparison with those producing IL-2 when the cells were stimulated by nominal Ag, by alloantigens, or by mitogens. One cycle of in vitro Ag restimulation and rest, however, enabled us to detect high proportions of IL-4-secreting cells among keyhole limpet hemocyanin-primed lymph node cells. With cultures set up at monoclonal cell doses, it was shown that IL-2 and IL-4 are secreted by separate precursor populations at this stage of their development. The IL-4-secreting cells were further shown to be dependent upon the presence of IL-2, either secreted by separate precursors or exogenously added, for the production of detectable amounts of IL-4. Analysis of the frequencies of helper cells producing both IL-2 and IL-4 at various stages of the in vivo immune response and the requirements for their growth and differentiation should give a better understanding of the relative contributions of each cell type.     

In vitro propagation of Agave arizonica Gentry & Weber

Agave arizonica Gentry & Weber, an extremely rare and endangered species native to Arizona, was successfully propagated in vitro using modified Murashige and Skoog media. Adventitious shoots developed from callus which formed on bulbil explants grown in a medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid. These shoots proliferated by subculture in media supplemented with 44.4 μM 6-benzylaminopurine, and either 0.5 or 5.4 μM naphthaleneacetic acid. Rooting occurred on shoots transferred to a growth regulator free medium. Rooted plants transferred to potting soil could be established under greenhouse conditions following gradual acclimatization indoors.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Gene order on the short arm of human chromosome 11: regional assignment of the LDH A gene distal to catalase in two translocations

Summary Leukemic cells with reciprocal translocations involving 11p13 and 14q13 were obtained from two patients with T-cell acute lymphoblastic leukemia and fused with mouse Ltk^- cells. DNA from independent hybrid clones was screened by Southern blot and hybridization to molecular probes for the human catalase and Ha-ras-1 genes. Several clones showed segregation of these two genes, indicating the presence of either the der 11 or der 14 human chromosomes. When DNA from these hybrid clones was examined for the presence of the human genes for calcitonin and γ-globin, both genes were found to segregate with the Ha-ras-1 gene and the der14 chromosome indicating that they lie distal to catalase. When the hybrid clones were examined for the presence of human lactate dehydrogenase A (LDH A) activity, only those clones containing the der14 chromosome expressed activity indicating that the LDH A gene is also distal to catalase on the short arm of chromosome 11.     

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.