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R E Hinkley

Publications and source records attributed to R E Hinkley.

At least 19 recordsLinked to original sources
PubMed1989

Selective identification of the paternal mitochondrion in living sea urchin eggs and embryos by chlorotetracycline.

When sea urchin eggs are pretreated with fluorescent chelate probe chlorotetracycline (CTC) and then fertilized with unlabeled sperm, a small, brightly fluorescent particle resembling the mitochondrion of free-swimming sperm both in size and fluorescent staining characteristics appears in the egg cytoplasm. This particle first appears near the base of the insemination cone and, like the paternal mitochondrion identified in previous ultrastructural studies, remains closely associated with the male pronucleus during its microtubule-dependent migration toward the egg center. These similarities strongly suggest that the fluorescent particle observed in the cytoplasm of living, CTC-pretreated sea urchin eggs is, in fact, the mitochondrion of the fertilizing sperm.

Animals↗
PubMed1988

The effects of inhalation anesthetics on calcium-stimulated exocytosis in a natural membrane model system.

Sea urchin egg cortices were used as an in vitro natural membrane model system to determine the effects of inhalation anesthetics on the Ca2+-regulated exocytotic fusion of cortical vesicles with the egg plasma membrane. When Ca2+ was either absent or present in amounts below the threshold for exocytosis, methoxyflurane, halothane, enflurane, isoflurane, chloroform and fluoroxene, at concentrations up to 5 mM, had no effect on the fusion of cortical vesicles with the plasma membrane. However, when Ca2+ was present at or above threshold levels for exocytosis, each of the tested anesthetics caused an inhibition of cortical vesicle fusion. Exocytosis was inhibited most effectively by methoxyflurane (55%), followed by halothane (30%), while fluoroxene consistently had the least effect (less than 5%). These observations support the view that volatile anesthetics can impair the Ca2+-regulated fusogenic activities of natural membranes and are consistent with other data showing that inhalational agents inhibit secretory processes in intact cells.

Anesthesia, Inhalation↗
PubMed1988

Student evaluation of accelerated program at the University of Miami.

The University of Miami School of Medicine has an accelerated, six-year B.S.-M.D. program called the Honors Program in Medicine (HPM). In 1985 the HPM and traditional program students were surveyed at the end of their first year in medical school regarding their opinions about accelerated medical education. Eighty-seven percent of the traditional students felt that HPM students had ability equal to that of the average medical student (that is, neither more nor less ability), but 41 percent felt that the HPM students were less mature than the average class member. The traditional students felt the HPM students were required to follow a rigid collegiate curriculum (76 percent) and had an inadequate nonscience background (68 percent) and that the HPM would negatively affect both the future professional competence of HPM participants (59 percent) and the medical profession (34 percent). A larger percentage of the HPM than the traditional students (63 versus 48 percent) reported earning more than a minor in nonscience subjects, and fewer HPM than traditional students (9 versus 36 percent) felt that pressure to maintain a high grade-point average limited their selection of college courses. The HPM students felt that they were well prepared for medical school academically (82 percent) and emotionally (91 percent). The HPM students excelled academically in college and in the preclinical curriculum.

Attitude↗
PubMed1986

Effects of the volatile anesthetic halothane on fertilization and early development in the sea urchin Lytechinus variegatus: evidence that abnormal development is due to polyspermy.

The volatile anesthetic halothane, when present at fertilization, dose-dependently increases the incidence of abnormally developing sea urchin embryos at the first cell division. Microscopic examinations of eggs stained with aceto-orcein or the DNA fluorochrome bisbenzimide and direct observations on isolated sperm aster complexes show that halothane induces polyspermy (multiple sperm entry) when present at fertilization. Experimental evidence suggests that anesthetic-induced polyspermy involves impairment of both the fast (electrically mediated) and slow (morphological) blocks to multiple sperm entry. These observations clearly show that relatively brief exposures to halothane at fertilization cause polyspermy and that this effect is almost certainly responsible for the ensuing abnormal development observed at the first cell division.

Animals↗
PubMed1986

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342.

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.

Animals↗
PubMed1986

Induction of the acrosome reaction in sea urchin spermatozoa by the volatile anesthetic halothane.

The volatile anesthetic halothane rapidly and dose-dependently induces the acrosome reaction in sperm of the sea urchin Lytechinus variegatus. The reaction occurs equally well in artificial sea water containing 10 mM Ca2+ and in low (less than 100 microM)-Ca2+ media. The anesthetic-induced acrosome reaction can be prevented by pretreating sperm with EGTA, lanthanum, or procaine. In contrast, the Ca2+ channel blockers D-600, verapamil, diltiazem, and nitrendipene do not prevent the reaction. Acrosomal processes induced by halothane are ultrastructurally identical to those induced by egg jelly, the natural inducer of the acrosome reaction. These results suggest that Ca2+ in low quantity is required for the anesthetic-induced acrosome reaction and may be derived from internal sources. Enflurane, isoflurane, and methoxyflurane also induce the acrosome reaction, indicating that volatile anesthetics may be useful in studying ion changes accompanying sperm activation.

Acrosome↗
PubMed1985

Isolation of intact sperm asters from fertilized sea urchin eggs.

We have developed a procedure for isolating intact sperm asters in quantity from fertilized sea urchin eggs. This procedure is based on detergent-extraction methods developed previously for the bulk isolation of mitotic apparatuses. Using this protocol it is possible to isolate sperm asters as soon as they appear in the fertilized egg or at any subsequent point in their brief existence.

Animals↗
PubMed1985

Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli.

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.

Actins↗
PubMed1985

Comparative effects of halothane, enflurane, and methoxyflurane on the incidence of abnormal development using sea urchin gametes as an in vitro model system.

The incidence of sea urchin embryos developing abnormally after their exposure to equimolar concentrations of halothane, enflurane, and methoxyflurane has been determined. Halothane concentrations in the 0.6-1.25 mM range caused 18-96% of the embryos to undergo abnormal cleavage at the first cell division. This is important because embryos exhibiting atypical cleavage patterns at the first cell division eventually involute and die before reaching gastrulation. Over the same range of concentrations, enflurane and methoxyflurane have minimal effects on development. However, when exposed to 2.5 mM methoxyflurane, nearly 40% of the cells did not fertilize. These results show that volatile anesthetic agents have decidedly different effects on development and suggest that the incidence of abnormal development may not correlate directly with the anesthetic potency of inhalational agents.

Abnormalities, Drug-Induced↗
PubMed1982

Structural changes in dividing sea-urchin eggs induced by the volatile anaesthetic halothane.

Fertilized Lytechinus eggs exposed to the volatile anaesthetic halothane before metaphase do not undergo cytoplasmic cleavage. This effect has been correlated with the failure of the contractile ring to assemble. Comparative studies on mitotic apparatuses isolated from control and halothane-treated cells show that halothane significantly impairs both spindle and aster growth as early as metaphase. When transferred to control solutions, halothane-treated cells initiate furrowing activity in association with either the first or second mitotic division, depending on the duration of the exposure to anaesthetic and the concentration employed. In contrast to these effects, halothane has no effect on any aspect of the cleavage process if applied later than metaphase. In this case, furrows develop even in the presence of halothane, deepen progressively, and complete cell division. These observations confirm and extend previous studies on echinoderm eggs exposed to volatile anaesthetics, and support the view that anaesthetic agents indirectly prevent cell cleavage by inhibiting the growth of mitotic apparatus.

Animals↗
PubMed1978

Macrotubules induced by halothane: in vitro assembly.

The formation of macrotubules by the volatile anaesthetic halothane was investigated in vitro using microtubule-enriched fractions of crayfish nerve cords. Sequential studies showed that macrotubules assemble from helical ribbons of 18-20 laterally associated microtubule protofilaments which fold upon themselves to form intact macrotubules averaging 48 nm in diameter. The initial rate of macrotubule assembly is dependent on the concentration of halothane employed and is stimulated by calcium. Glycerol pretreatment blocked macrotubule formation by halothane and caused preformed macrotubules to reassemble rapidly into typical microtubules. These experiments show that microtubules and macrotubules require different conditions for assembly and support the contention that macrotubule formation by halothane is due to a direct interaction between the anaesthetic molecule and the microtubule subunit.

Animals↗
PubMed1977

Cultured neuroblastoma cells and halothane: effects on cell growth and macromolecular synthesis.

Cultured mouse neuroblastoma cells were grown in air-CO2 or air-CO2-halothane-gassed incubators. In the presence of halothane the growth rate of the cells was inhibited in a dose-dependent manner; 2 per cent halothane completely inhibited cell growth, while at 0.3 per cent halothane, the growth rate was 74 per cent of the control rate. The biosynthesis of protein and RNA in cells grown in the control atmosphere and that in cells grown in 1 per cent halothane were compared by several techniques. No significant difference between the rates of synthesis of these two macromolecules could be detected. Furthermore, a comparison of labeled protein and RNA by SDS-polyacrylamide gel electrophoresis revealed no qualitative difference. From this and previous work it is concluded that halothane affects the morphology and growth rate of cultured mouse neuroblastoma cells by disrupting cytoplasmic actin-like micro-filaments.

Animals↗
PubMed1975

Tannic acid-stained microtubules with 12, 13, and 15 protofilaments.

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.

Animals↗