Correlation of conformational changes in the acrosome stabilizing factor (ASF) with its biological activity

The rabbit Acrosome Stabilizing Factor (ASF) is a glycoprotein synthesized in the corpus epididymis that reversibly decapacitates sperm. The effects of altering the conformation of ASF were evaluated by using a competitive enzyme-linked immunoabsorption assay (ELISA) with monoclonal antibodies that recognized either sequential or conformational determinants and/or an in vivo decapacitation assay. Heat denaturation (80 degrees C for 30 min) of affinity-purified ASF resulted in destruction of its native conformation concurrent with its loss of biological activity. Acid pH treatment of ASF also resulted in a conformational change in ASF, which caused a shift from the dimeric form (MW = 260,000) to the monomeric form (MW = 130,000). This manipulation allowed the biological activity of the monomeric form of ASF to be assayed separately from the dimer. The monomer was found to be biologically inactive. Proteolysis with trypsin or Staphylococcus-V8 treatment resulted in loss of the native conformation of the molecule, but Staphylococcus-V8 did not destroy the sequential determinant recognized in this analysis. This work indicates that conformation of the ASF macromolecule is important for its biological activity, and also provides a rapid means to evaluate potential decapacitation activity of purified ASF.     

Enthalpy and entropy changes for the intercalation of small molecules to DNA. II. Ethidium and propidium fluoride

Enthalpy changes (ΔH_B) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na⁺ ion concentrations of 0.019, ΔH_B = ?8.3 and ?7.9 ± 0.3 kcal mol^?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔH_B is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔH_B values for propidium did not depend appreciably on the Na⁺ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔG_B values for the binding of these cations to DNA, we found the variation of ΔS_B with Na⁺ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na⁺ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.     

Shape effects in elastic scattering by orientationally random ensembles of simple model viruses

Recent theoretical work has shown that the complete set of polarized elastic light scattering studies should yield information about particle structure that has so far hardly been utilized. We present calculations of such light-scattering properties for a number of model structures, exploring particularly the size limit at which the new effects should become visible. The particles are assumed to be randomly oriented in aqueous suspension, and all identical to each other. We compare several particle models of differing geometrical shape, but with identical forward scattering power and identical radii of gyration. We find that one of the ten observables shows particularly desirable properties as a general large-particle characterization parameter: it is nonzero for all structures, it approaches zero as particle size decreases, and it shows an angular dependence that distinguishes among models of different shape. Assuming incident light at 350 nm, it differentiates between different shaped particles with radii of gyration as small as 50 nm. Such particles are well below the optical resolution limit and about the size of many types of viruses.     

Tip size of ion-exchanger based K+-selective microelectrodes. I. Effects on selectivity

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to study the dependency of ion selectivity upon tip size. The selectivity of K+ ISMs depended on tip size and barrel configuration. Within the range of tip diameters tested (approximately 0.5-6 micron) all K+ ISMs constructed of two barrels glued side by side ("figure-eight glass") exhibited sensitivity to K+ and NH4+. Figure-eight K+ ISMs with tip diameters less than 1.5 micron were not sensitive to tetramethylammonium, tetraethylammonium, or choline, whereas K+ ISMs with tip diameters greater than or equal to 1.5 micron sensed all of the quaternary amines. Tip size dependent selectivity was not present in K+ ISMs made from thick septum theta glass. The explanation for tip size dependent changes in ion selectivity is unknown but a discussion of theoretical possibilities is given.     

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex. Two enzymes (side-chain cleavage and 11 beta-hydroxylase) are co-localized in the same mitochondria

In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.     

Nuclear DNA analysis of koilocytic and premalignant lesions of the uterine cervix.

Cervical biopsy samples were taken from 79 patients who had various grades of cervical intraepithelial neoplasia or who showed evidence, in the form of koilocytosis, of human papillomavirus infection of the uterine cervix and from 10 women with normal cervices. The DNA content of the cells in the samples was analysed by flow cytometry. Analysis of the data obtained showed that the biopsy samples from women with cervical intraepithelial neoplasia and human papillomavirus lesions contained significantly more dividing cells (31.2% of cells from human papillomavirus lesions with no cervical intraepithelial neoplasia and 33.06%, 29.89%, and 31.76% of cells from cervical intraepithelial neoplasia grades I, II, and III, respectively) than those from women with normal cervices (21.6%). The proportion of aneuploid samples from the group who showed evidence of human papillomavirus infection only (18.2%) did not differ significantly from the group with cervical intraepithelial neoplasia grade III (21.2%). Aneuploidy and an increased rate of cellular proliferation are recognised characteristics of malignancy. These results therefore support the view that human papillomavirus plays an important part in the aetiology of cervical carcinoma and are relevant to the clinical management of patients.     

List sizes and use of time in general practice.

The claim that list sizes in general practice should continue to fall towards a national average of 1700 patients rests heavily on the assumption that the extra time available to doctors would be used mainly for longer consultations, resulting in better standards of care. Evidence suggests, however, that the time is more likely to be used to increase rates of consultation in surgeries and home visits and to reduce the length of the working week. A national, random sample of 2104 principals in general practice in England and Wales were questioned about their allocation and use of time. The response rate was 67%, and no large biases in response were detected. The smaller their personal list size the less time general practitioners spent on all aspects of their work and the higher their rates of consultation and home visiting. The effects of further reductions in list sizes would be haphazard, being differentially distributed across the range of list sizes. Longer consultations would probably result, but most of the extra time would probably be used in higher rates of consultation in surgeries and home visits and some would be taken as free time.     

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.     

The evolutionary importance of mechanoreception in three erpobdellid leech species

Summary The ability of an erpobdellid leech, Erpobdella montezuma, to discriminate between two congeneric amphipod prey (Hyalella montezuma and Hyalella azteca) through mechanoreception was examined. Both E. montezuma and H. montezuma, a pelagic filter feeder, are endemic to the near thermally constant environment of Montezuma Well, Arizona, and form a highly specialized predator-prey relationship. In contrast, H. azteca, a benthic detritivore, is widely distributed throughout North America. Erpobdella montezuma was highly responsive to the prey signals of the endemic H. montezuma and showed only a limited response to H. azteca. An inverse relationship occurred between the frequency of attack and size of the leech. The abilities of Erpobdella punctata and Nephelopsis obscura, neither found in Montezuma Well, to detect the signals of both amphipod prey were also examined. These species with non-specialized diets showed a low response to the signals of both prey species. This study demonstrates that very close predator-prey relationships can develop over evolutionary time in isolated aquatic systems through the use of mechanoreception.