Stabilization of DNA triple helices by a series of mono- and disubstituted amidoanthraquinones.

We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base-functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7-disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5-disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure-activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.     

An aerodynamic valve in the avian primary bronchus.

The segmentum accelerans in geese is a constriction in the caudal end of the primary bronchus. Experimental evidence suggests that this part of the airway functions as an inspiratory aerodynamic valve, accelerating the incoming airstream past the ventrobronchial openings. The luminal diameter of the segmentum accelerans dilates in the presence of elevated CO2 levels, probably through relaxation of smooth muscle. Physiological control of the segmentum accelerans may permit inspiratory aerodynamic valving to be maintained throughout a wide range of ventilatory flows.     

Form of inorganic carbon involved as a product and as an inhibitor of c(4) Acid decarboxylases operating in c(4) photosynthesis

These studies demonstrated that CO₂ rather than HCO₃⁻ is the inorganic carbon metabolite produced by the C₄ acid decarboxylases involved in C₄ photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate [PEP] carboxykinase). The effect of varying CO₂ or HCO₃⁻ as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K_mCO₂ was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO₂ concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HCO₃⁻. For NADP malic enzyme CO₂ was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO₂ and HCO₃⁻.     

A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Allelic Variation within the Emv-15 Locus Defines Genomic Sequences Closely Linked to the agouti Locus on Mouse Chromosome 2

Gene(s) at the agouti locus act within the microenvironment of the hair follicle to switch pigment synthesis in the melanocyte between eumelanin (black or brown pigment) and phaeomelanin (yellow pigment). Many phenotypic variants of this locus have been described. The mechanism(s) of gene action causing such variation in coat-color phenotype is not known. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to the lethal yellow mutation of the agouti locus provides a means to molecularly access genes at or near the agouti locus. We have identified and used a unique mouse sequence flanking the Emv-15 provirus to define three alleles of the Emv-15 locus. We found a correlation between the presence of specific Emv-15 alleles and the origins of specific agouti locus mutations, confirming close linkage. However, we found some exceptions which suggest that the Emv-15 locus is closely linked to, but genetically separable from, the agouti locus.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Application of a new method for detecting the phenotype of target binding cells

Summary A new method was developed for detecting the phenotype of target binding cells (TBC) in a single-cell assay system. This methodology was evaluated during a clinical trial of recombinant interferon alfa-2a (rIFN alfa-2a) for the treatment of 10 metastatic renal cell carcinoma patients. Total TBC with K562 targets, HNK-1+ TBC, and HLA-DR+ TBC were quantitated during rIFN alfa-2a therapy. A significantly increased proportion of lymphocytes bound to target cells on day 9 of therapy bore the HNK-1 marker. This proportion subsequently declined to pretreatment levels. Total TBC paralleled the rise and fall in HNK-1+ TBC. HLA-DR+ TBC binding to targets remained constant and low throughout therapy. These findings suggest that rIFN alfa-2a early in therapy (day 9) caused the recruitment of additional HNK-1+ cells into binders. However, with continued therapy, this proportion reverts to pretreatment levels. The results of this clinical trial served to illustrate the ability of the modified single-cell assay system to detect TBC phenotype.Supported in part by Hoffman-La Roche, NIH grant CA 12582, and Jonsson Comprehensive Cancer Center grant CA 15866Dr. Figlin is a recipient of an American Cancer Society Junior Faculty Fellowship-JFCF 762-A     

Quantitative DNA analysis of low grade cervical intraepithelial neoplasia and human papillomavirus infection by static and flow cytometry.

Quantitative deoxyribonucleic acid (DNA) analysis of cervical biopsy specimens from 26 women with cytological, colposcopic, and histological evidence of mild cervical atypia consistent with cervical intraepithelial neoplasia grade I, reactive atypia, or human papillomavirus infection alone or in combination was performed in a comparative evaluation of Feulgen microspectrophotometry, the fast interval processor image analysis system, and flow cytometry. The fast interval processor image analysis system showed a distinct advantage over the other methods, being faster and allowing the operator to see the cells that were selected for measurement. The three methods of measurement together showed that the DNA content of at least 2% of the cells measured exceeded 5C (C being the haploid amount of DNA in a normal cell and 2C representing the diploid complement of a normal cell) in all cases of cervical intraepithelial neoplasia grade I and reactive atypia and in 87% of those reported as showing human papillomavirus infection alone. In contrast, the DNA content of cervical biopsy specimens from the transformation zone of 11 normal controls did not exceed 4C. This study shows the value of using a DNA threshold--that is, the "5C exceeding rate"--to distinguish between normal and neoplastic appearances of the cervix. These results support the view that cervical infection by human papillomavirus is a true precursor of neoplasia.