Estimating the recombination frequency for the MN and the Ss loci

Linkage analysis of 146 informative families for MN and Ss resulted in an estimate of the recombination frequency greater than previously reported. Our total is 7 recombinant children out of 467 individuals, including 1 confirmed recombinant (retested and HLA-compatible) and 6 not verified. The 95% confidence interval of our estimate of recombination is 0.0033-0.0167. Our results are compared with two earlier studies.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.     

2,4-D-Induced Changes in the K+ Uptake of Wheat Roots at Different pH Values

The effects of an auxin herbicide, 2,4-D, at a concentration of 0.01 mM, on the K⁺ uptake and efflux of excised roots of wheat (Triticum aestivum L. cv. Rannaya) were investigated at different pH values. The K⁺ movement was monitored with a K⁺ (⁸⁶Rb) tracer. In parallel experiments the ATPase activities of microsomal fractions were determined by the inorganic phosphate liberation method. 2,4-D inhibited the K⁺ uptake especially at low pH, irrespective of whether Ca²⁺ was present or not. No marked changes were observed in the K⁺ efflux properties at pH values above 4. The inhibitory effect on K⁺ uptake exhibited a correlation with the hydrocarbon solubility of the herbicide, but not with the 2,4-D-induced decrease of the ATPase activity. It is suggested that 2,4-D exerts a non-specific effect on the lipid-protein interactions, giving rise to a generalized alteration of the transport barrier properties of the plasma membrane even at as low a concentration as 0.01 mM.     

The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells

HP-1 is a 30-residue cysteine- and arginine-rich peptide of the human neutrophil primary granule and is the most abundant human representative of the family of peptides variously called defensins and corticostatins. Peptides belonging to this family have many biological activities including the non-oxidative destruction of ingested microorganisms, the inhibition of adrenocorticotropin-stimulated synthesis of glucocorticoids, monocyte chemotaxis, the non-cytolytic inhibition of [3H]thymidine incorporation in HL-60 promyelocyte-like cells and the stimulation of nifedipine-sensitive calcium channels. Using a combination of reversed-phase and size-exclusion high performance liquid chromatography and an HP-1 radio-immunoassay, three immunoreactive peptides were detected and isolated from the promyelocyte-like cell line, HL-60, and from leukocytes of patients with chronic myelogenous and chronic lymphocytic leukemias. One of these peptides was HP-1 itself. A second was identified by gas-phase Edman microsequencing as an amino-terminally extended fragment of the HP-1 precursor which we call HP1-56. The third is likely to arise from enzymatic cleavage of the precursor at a dibasic site. Of the leukemic cells the greatest amount of HP1-56 relative to HP-1 was found in cells from a patient in myeloblastic crisis but overall the richest source of HP1-56 relative to HP-1 was found to be in fetal lung tissue. HP1-56 is difficult to detect in normal peripheral neutrophils and its presence in cells that are actively biosynthesizing primary granule components such as HL-60 may make it useful for studying the biosynthesis of granule polypeptides, their ontogeny, and possibly as a marker protein for leukemic diseases.     

Corticostatic peptides.

In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.     

N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.     

Metabolism of polyadenylic acid sequences during germination of blastocladiella emersoni: Zoospores.

The metabolism of the poly(A) sequences isolated from Blastocladiella emersonii was followed during the first hour of germination. Poly (A) sequences synthesized during the first 30 min of germination do not undergo detectable changes in size. During the first 45 min of germination, poly(A) sequences synthesized during zoosporogenesis decrease in size to the extent that there is essentially no size overlap between poly(A) fragments which were present in the zoospore and newly synthesized poly(A) sequences. The results presented indicate that during germination, polyadenylation occurs in RNA molecules which were present in the zoospore but lacked poly(A) sequences. No detectable size differences were observed between poly(A) sequences added to newly synthesized RNA compared to those added to the nonpolyadenylated RNA present in the zoospore during germination. Cycloheximide did not prevent the observed decrease in size of the poly(A) sequences during germination.