Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease

A scanning proton microprobe has been used for the elemental microanalysis of individual fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. The cells were cultured on a thin ultra-clean nylon foil and retained on that surface for analysis. The focused high-energy proton beam was used to irradiate selected individual cells and elemental information was derived from X-ray and backscattered proton data. The sensitivity of the scanning proton microprobe to trace concentrations of heavy elements has allowed this elemental information to be used to identify individual cells as being either normal or a Menkes' mutant. The cell identification was based on the application of discriminate analysis to a data set formed from the ratios of copper to each of the macroelements present in the cell. This method of cell identification offers the promise of rapid diagnosis of Menkes' disease.     

Gravitropically-stimulated seedlings show autotropism in weightlessness

In a spaceflight experiment, autotropism by oat ( Avena sativa L.) coleoptiles following gravitropic responses was prominent in weightlessness: counter-reactions led to the straightening of the curved coleoptiles. This was not the case during clinorotation on earth. The autotropic reactions appeared to be related to the stimulus received during the stimulus period, i.e. the greater the response the greater the autotropic counter-reaction. Previous models of the gravitropic system which predicted that coleoptiles would not straighten in weightlessness are disproved. A modification to one of the models is proposed which includes the autotropic response observed in spaceflight. The nature of the counter-reactions in the absence of gravitropic stimulation is discussed.     

The efficacy of CGS 20267 in suppressing estrogen biosynthesis in patients with advanced stage breast cancer

The pharmacologic inhibition of aromatase activity has been the focus of clinical trials in patients with advanced stage breast cancer. Recent developments with imidazole compounds that inhibit aromatase activity suggest their clinical use as potent inhibitors of estrogen biosynthesis in postmenopausal breast cancer patients. In this Phase I, open-label, dose-range finding study, we examined the inhibitory potency of CGS 20267 on blood and urine levels of estradiol, estrone and estrone sulfate in 8 patients with metastatic breast cancer. Studies included evaluation of adrenal and thyroid function to look for evidence of general hydroxylase inhibition at dose levels effective for aromatase blockade. Patients were administered CGS 20267 at doses of 0.1 and 0.25 mg, once a day in ascending doses over a 12-week period. Preliminary data reveal that CGS 20267 elicits a striking suppression in plasma estradiol, estrone and estrone sulphate which was observed in some patients as quickly as within 24 h of the first dose. Estrogen suppression of over 90% was achieved within 2 weeks of therapy. No alterations in either baseline or ACTH (cortrosyn) stimulated cortisol and aldosterone levels were observed through the 12 weeks of therapy. In addition, 24 h urine sodium and potassium values were not appreciably altered during therapy. We conclude that CGS 20267 is a potent, specific inhibitor of estrogen biosynthesis in postmenopausal patients with metastatic breast cancer and effectively reduces blood and urine estrogens to undetectable levels.     

Binding of protein chemotactic factors to the surfaces of neutrophil leukocytes and its modification with lipid-specific bacterial toxins

Summary The binding to neutrophil leukocytes of human serum albumin (HSA), which is chemokinetic for leukocytes, i.e. influences their rate of locomotion, and of alkali-denatured HSA, which is chemotactic for leukocytes, i.e. influences their direction of locomotion, was studied. Native serum albumin showed low affinity binding to the neutrophil surface. Denatured serum albumin showed saturable binding with a K_a of approximately 10⁶ litres per mole to about 10⁶ binding sites per cell. Another protein chemotactic factor, _s-casein, gave similar binding. These results exclude that chemotactic reactions to denatured proteins are mediated in a completely non-specific manner and suggest the presence on the cell of a restricted number of defined recognition sites. Binding was reduced following treatment of the cells with either of two lipid-specific bacterial toxins, perfringolysin, the -toxin of Clostridium perfringens, an oxygen-labile cholesterol-specific toxin, and Staphylococcus aureus Sphingomyelinase C. Both have previously been shown to reduce chemotactic reactions and both were used at doses which did not reduce cell viability. These results suggest an important, and possibly direct, role for membrane lipid in the binding sites for chemotactic factors. Visual analysis of the behaviour of perfringolysin-treated neutrophils showed that these cells were still capable of chemotactic locomotion. The cells appeared to be less efficient than normal in detecting chemotactic gradients only when at a distance from the gradient source, a finding which is consistent with reduced binding of the chemotactic factor to the cell surface.     

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome.

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.     

Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence.

We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.     

Depsidone constituents from the quintaria group of Nephroma species

A new lichen depsidone was isolated, in the form of its triacetate derivative from the acetylated extracts of Nephroma antarcticum and has been demonstrated to be hypoconstictic acid-triacetate. Two related depsidones, hypostictic acid hyposalazinic acid, were isolated from N. australe.     

Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes,granulocytes, lymphocytes,and platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10⁷ cells, granulocytes have 17–201 units/10⁷ cells, lymphocytes have 91–462 units/10⁷ cells, and platelets have 1.1–7.1 units/10⁷ platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.This work was supported by funds allocated by the Agricultural Experiment Station, Michigan State University. Michigan Agricultural Experiment Station Journal No. 8727.