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Biomedical subjects

R A Cone

Publications and source records attributed to R A Cone.

At least 37 records · Page 2Linked to original sources
PubMed1993

Nonoxynol-9 protects mice against vaginal transmission of genital herpes infections.

A vaginal application of a commercially available contraceptive jelly containing nonoxynol-9 (N9) prevented vaginal transmission of herpes simplex virus type 2 (HSV-2) infections in the mouse. When N9 jelly was delivered to the vagina with the virus inoculum, 20 s before inoculum, or 5 min before inoculum, mice were completely protected from visible infection (P < .001). Protection lasted for at least 30 min (P < .03), and significant protection occurred even when the N9 jelly was delivered 15 min after the HSV-2 inoculum (P < .05). These results are consistent with results of studies using N9 products in other animal models and suggest that N9-based contraceptive products can provide significant protection against vaginal transmission of enveloped virus infections in animals.

Animals↗
PubMed1993

The cat/feline immunodeficiency virus model for transmucosal transmission of AIDS: nonoxynol-9 contraceptive jelly blocks transmission by an infected cell inoculum.

OBJECTIVES: To develop an animal model to study transmucosal lentivirus transmission, and to determine whether topical application of contraceptive jelly can block transmission by an infected cell incoulum. DESIGN: Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an AIDS-like disease in domestic cats. HIV is transmitted primarily across mucosal surfaces, and infected cells may be important in this transmission. We tested the ability of FIV-infected cells to transmit infection across the vaginal, rectal and oral mucosa of the cat, and whether a vaginal contraceptive jelly could prevent such transmission. METHODS: An inoculum consisting of 2 million FIV-infected primary cat T cells was administered vaginally, rectally or orally to female cats that had received either no pretreatment or pretreatment with a contraceptive jelly containing the detergent nonoxynol-9 as spermicide. Transmission was detected by monitoring recipient animals for viral antibodies and by viral cultures of blood leukocytes. RESULTS: A single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina or rectum (10 out of 11 animals became infected). Pretreatment of the vagina (five animals) or rectum (four animals) with contraceptive jelly protected all animals from transmission by the highly infectious inoculum. CONCLUSIONS: The cat/FIV model provides an efficient means to study transmucosal transmission of lentivirus infections, and for assessing vaginal barrier methods that could block transmission. One such method, nonoxynol-9 contraceptive jelly, effectively prevents transmucosal transmission by an FIV-infected cell inoculum.

Animals↗
PubMed1992

Controlled vaginal delivery of antibodies in the mouse.

Controlled delivery of monoclonal antibodies to the mucus secretions of the vagina might provide women with passive immunoprotection against both sexually transmitted diseases and unwanted pregnancy. We have developed intravaginal devices composed of poly(ethylene-co-vinyl acetate) (EVAc) that continuously release IgG antibodies for over 30 days into buffered saline, and we have tested these devices in the vagina of mice. Polymeric devices containing either BSA (as a test reagent for proteins) or anti-hCG antibody, when inserted into the vaginas of mice, provided a continuous supply of either BSA or hCG-binding antibodies to the vaginal mucus for 30 days. Antibodies released by the devices achieved high concentration in the mucus within the lumen of the vagina, but did not significantly ascend into the uterine horns, as determined by epifluorescence microscopy of fluorescently labeled mouse IgG and by immunohistochemical localization of rabbit IgG. Our results suggest that long-term intravaginal delivery of functionally intact antibodies can be achieved with devices composed of EVAc.

Administration, Intravaginal↗
PubMed1990

The strength of non-covalent biological bonds and adhesions by multiple independent bonds.

Non-covalent bonds of the same type as antibody-antigen bonds--"affinity bonds"--are one of the most important types of structural bonds on the cellular level, mediating interactions such as cell-cell and cell-extracellular matrix adhesion, providing the integrity of such structures as microtubules and actin filaments, and producing force in muscle, flagella and cilia. The affinity bonds in these and many other interactions are subject to forces during the life of the cell. However, the tensile strength of these bonds, and how this determines the strength of the interactions on the cellular level, is largely unknown. We have attempted to calculate the tensile strength of an affinity bond by modelling binding as a multistep process in which the binding molecules first diffuse together, overcome an activation energy, and then bind. By calculating the energies associated with each step, the energy actually contained in the bond can be determined, yielding the bond strength. The "average" affinity bond is thus found to have a tensile strength of about 40 mu dyn. However, the temporal nature of affinity bonds (thermal fluctuations will eventually break them) causes the strength of the interactions to be actually somewhat weaker, with the outcome of putting tension on a group of bonds depending strongly on the conditions.

Animals↗
PubMed1990

Macromolecules released from polymers: diffusion into unstirred fluids.

Polymers that release macromolecules may be useful for preventing and treating human disease. In certain applications of polymeric controlled release, like drug therapy of brain disease and immunoprotection of mucus epithelia, effectiveness may be limited by diffusion through an unstirred fluid near the polymer. Using computer-assisted epifluorescence microscopy, we have examined the local distribution of fluorescently labelled macromolecules released from an ethylene-vinyl acetate copolymer matrix into unstirred layers of phosphate-buffered water and mid-cycle human cervical mucus. Diffusion coefficients in the fluid were determined by observing the concentration profiles as a function of time. Diffusion coefficients determined for fluorescein, bovine serum albumin, and three classes of human immunoglobulins (IgG, sIgA and IgM) in phosphate-buffered water were in good agreement with literature values. For fluorescein, albumin and IgG, diffusion in mucus was comparable with diffusion in water: the largest molecule tested was slowed by only a factor of 3.

Biocompatible Materials↗
PubMed1990

Dense fibers protect mammalian sperm against damage.

The relative tensile strengths of the sperm of seven mammalian species and sea urchins have been measured by determining the minimum shear necessary to kill them (assayed by lack of motility) when they are suspended in a viscous fluid. In general, long sperm are killed by smaller shears than short sperm. However, the longer sperm are not as fragile as would be expected from theoretical predictions. Their additional tensile strength correlates well with the size of their dense fibers; a theory that includes the dense fiber contributions accurately predicts the sperm tensile strength for most of the species in which this has been measured. This added strength may be necessary to protect sperm from shear forces encountered during epididymal transport and especially during ejaculation, as these forces are strong enough to kill long sperm if they are not strengthened.

Animals↗
PubMed1988

Mechanics of sperm-egg interaction at the zona pellucida.

Mammalian sperm traverse several layers of egg vestments before fertilization can occur. The innermost vestment, the zona pellucida, is a glycoprotein shell, which captures and tethers the sperm before they penetrate it. We report here direct measurements of the force required to tether a motile human sperm as well as independent calculations of this force using flagellar beat parameters observed for sperm of several species on their homologous zonae. We have compared these sperm-generated forces with the calculated tensile strength of sperm-zona bonds, and found that a motile sperm can be tethered, at least temporarily, by a single bond. Therefore, sperm can be captured by the first bond formed and tethered permanently by a few. The sperm cannot subsequently penetrate the zona unless the bonds are first eliminated. However, premature elimination would simply allow the sperm to escape. Therefore, not only must the bonds be eliminated, but the timing of this must be regulated so that the sperm is already oriented toward the egg and beginning to penetrate as the bonds are broken.

Animals↗
PubMed1986

Mechanical immobilization of rat sperm does not change their oxygen consumption rate.

Rat sperm are mechanically immobilized by the mucin immobilin during storage in the cauda epididymidis. We thought this mechanical immobilization might lower the respiration rate of caudal sperm since it is well established that both the respiration rate and adenosine triphosphate (ATP) dephosphorylation rate of certain invertebrate sperm are markedly reduced when they are mechanically immobilized. To measure the oxygen consumption rates of rat sperm, we used a conventional polarographic oxygen sensor. However, we used an unstirred sample chamber since we found that sperm are unusually fragile and are quickly killed by even moderate stirring. When caudal sperm were diluted in physiological salt solutions, they became vigorously motile and consumed oxygen at a rate of approximately 20 microliter O2/10(8) sperm/hour at 34.5 degrees C for dilutions between 20- and 200-fold. In confirmation of the work of Brokaw et al., we found that when the motility of sea urchin sperm was reduced progressively with increasing amounts of methylcellulose, the sperm consumed oxygen at a markedly reduced rate. In contrast, we found that when caudal rat sperm were mechanically immobilized, either by methylcellulose or by immobilin, their oxygen consumption rate did not differ detectably from that of vigorously motile sperm. Thus, the coupling of metabolism to motility differs significantly between these two types of sperm. We conclude that immobilin does not change the respiration rate of caudal rat sperm.

Animals↗
PubMed1985

Transient dichroism in photoreceptor membranes indicates that stable oligomers of rhodopsin do not form during excitation.

If a photoexcited rhodopsin molecule initiates the formation of rhodopsin oligomers during the process of visual excitation, the rate of rotational diffusion of the rhodopsin molecules involved should change markedly. Using microsecond-flash photometry, we have observed the rotational diffusion of rhodopsin throughout the time period of visual excitation and found that no detectable change occurs in its rotational diffusion rate. Partial chemical cross-linking of the retina yields oligomers of rhodopsin and causes a significant decrease in the rotational diffusion rate of rhodopsin even when as little as 20% of rhodopsin is dimeric. Moreover, the pattern of oligomers formed by cross-linking, taken together with the magnitude of decreases in rotational diffusion rate accompanying the cross-linking reaction, suggests that rhodopsin is a monomer in the dark-adapted state. The experiments reported here show that photoexcited rhodopsin molecules do not irreversibly associate with unbleached neighbors during the time course of the receptor response. Hence, it is not likely that stable oligomers of rhodopsin trigger the excitation of the photoreceptor cell.

Animals↗
PubMed1984

Light activates rotations of bacteriorhodopsin in the purple membrane.

To investigate how a photoactivated chromophore drives the proton pump mechanism of bacteriorhodopsin, we have observed how the chromophore rotates during the photocyle. To do this, we examined the dichroism induced in aqueous suspensions of purple membrane fragments by flashes of linearly polarized light. We find that the flash stimulates both the photocycling chromophores and their noncycling neighbors to undergo large (greater than 10 degrees - 20 degrees) rotations within the membrane during the photocycle, and that these two chromophore populations undergo distinctly different sequences of rotations. All these rotations could be eliminated by glutaraldehyde fixation as well as by embedding unfixed fragments in polyacrylamide or agarose gels. Thus, in these immbolizing preparations the chromophore can photocycle without rotating inside a bacteriorhodopsin monomer by more than our detection limit of 2 degrees - 5 degrees. The large rotations we observed in aqueous suspensions of purple membranes were probably due to rotations of entire protein monomers. The process by which a photocycling monomer causes its noncycling neighbors to rotate may help explain the highly cooperative behavior bacteriorhodopsin exhibits when it is aggregated into crystalline arrays of trimers.

Bacteriorhodopsins↗
PubMed1983

Rat sperm are mechanically immobilized in the caudal epididymis by "immobilin," a high molecular weight glycoprotein.

In many mammals, sperm are immotile while stored in the caudal epididymis and do not become motile until ejaculation. We report here our investigation of the mechanism that initiates motility in mature rat epididymal sperm. We found that an external "activator" is not required to initiate rat sperm motility since immotile sperm started to swim immediately when exposed to solutions that contributed only osmotic support. Instead, we found that epididymal rat sperm are kept fully immobilized by a high molecular weight glycoprotein, "immobilin," that we have isolated from rat cauda epididymal (CE) fluid. Our results strongly support the hypothesis that immobilin inhibits sperm motility mechanically simply by creating a highly viscoelastic environment: 1) rat CE fluid inhibited the motility of such disparate cells as rat sperm, E. coli. and rabbit sperm (which are fully motile in rabbit CE fluid), 2) the degree to which a variety of enzymatic treatments or slight dilution of the CE fluid initiated sperm motility depended only on the degree to which the treatment reduced the viscoelastic drag of the fluid, and 3) centrifugation of CE fluid simultaneously copurified the component of the fluid that immobilizes the sperm, the component that renders the fluid viscoelastic, and the glycoprotein immobilin.

Animals↗
PubMed1981

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.

Animals↗
PubMed1978

Bacteriorhodopsin induces a light-scattering change in Halobacterium halobium.

When suspensions of Halobacterium halobium are exposed to bright light, the light-scattering properties of the bacteria change. This light-scattering response can produce a transmission decrease of about 1% throughout the red and near-infrared region. The action spectrum for the light-scattering response appropriately matches the absorption spectrum of bacteriorhodopsin. The response is eliminated by cyanide p-trifluoro-methoxyphenylhydrazone, a proton ionophore, and by triphenylmethylphosphonium, a membrane permanent cation. A mild hypertonic shock induces a similar light-scattering change, suggesting that bright light causes the bacteria to shrink about 1% in volume, thereby producing the light-scattering response.

Adaptation, Physiological↗
PubMed1978

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.

Animals↗
PubMed1975

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.

Animals↗