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Biomedical subjects

M J Dimino

Publications and source records attributed to M J Dimino.

At least 19 recordsLinked to original sources

Educating medical students for Alaska.

Because Alaska does not have its own medical school, it has become part of WAMI (Washington, Alaska, Montana, Idaho), an educational agreement with the University of Washington School of Medicine (UWSM). Each year, 10 Alaskans are accepted into the entering class of UWSM and spend their first year at the University of Alaska Anchorage (UAA). UWSM third- and fourth-year medical students can obtain some of their clinical experience in Alaska. To meet the needs of Alaska, students are chosen based on academic and personal records, as well as the likelihood of their returning to Alaska for practice. To this end, over the last seven years 30% of accepted students have come from rural communities and 10% are Alaska Natives. The curriculum for the first year includes several sessions dedicated to Alaska health problems, cross-cultural issues, and Alaska's unique rural health care delivery system. Students do two preceptorships--one with a private primary care physician and one with a physician at the Alaska Native Medical Center. Additionally, students have the option to spend a week at a rural site to learn about the community's health care system. An Alaska track is being developed whereby an Alaskan UWSM student can do most of the third year in state via clerkships in family medicine, obstetrics/gynecology, psychiatry, internal medicine, and pediatrics. All UWSM students at the end of their first year can elect to participate for one month in the R/UOP (Rural/Underserved Opportunities Program), which includes several Alaska sites. The overall goals of these approaches are to educate UWSM students, especially Alaskans, about the state's health needs and health care system and to encourage UWSM graduates to practice in the state.

Alaska↗

Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs.

In addition to increasing cyclic adenosine 3',5'-monophosphate (cAMP) levels, luteinizing hormone (LH) stimulation of granulosa results in phosphoinositide hydrolysis producing inositol trisphosphate (IP3) and diacylglycerol. The roles of these putative second messengers were investigated by measuring production of progesterone and inositol phosphates by granulosa from medium-sized porcine follicles (3-7 mm) after 15 min incubation with or without LH (1 microgram/ml), 5 microM dibutyryl cAMP (dbcAMP), or 5 microM 1-oleoyl,2-acetylglycerol (OAG). Compared to a control rate of 5.4 pmoles/10(7) cells/15 min, LH and dbcAMP stimulated progesterone production to 12.8 and 15.9 pmoles, respectively, and OAG decreased progesterone production to 3.7 pmoles. LH also stimulated inositol phosphate (IP) and bisphosphate (IP2) accumulations by approximately 5-fold and IP3 accumulation by 20-fold. In experiments where granulosa were premeabilized with saponin, LH, dbcAMP, and IP3 stimulated progesterone production from 1.3 pmol in control cells to 5.2, 3.2, and 5.1 pmol, respectively, and OAG decreased progesterone production to 1.0 pmol. LH stimulated accumulation of all inositol phosphates in permeabilized cells, whereas the addition of IP3 only increased IP2 and IP3 accumulations. In granulosa preincubated with 0.9 mM [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, A23187 increased progesterone production from 3.7 to 5.8 pmol. Addition of 1-20 nmoles IP3 to 10(7) granulosa incubated in a Ca2+-free medium increased Ca2+ efflux linearly. These data suggest that IP3 may have a role in regulating steroid production in granulosa by regulating intracellular Ca2+.

Animals↗

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone.

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.

Animals↗

Early effects of luteinizing hormone on mitochondrial phosphoinositides in ovarian follicles.

It is not clear if luteinizing hormone (LH) stimulates breakdown as well as synthesis of phosphoinositides in ovarian tissue. Possibly, LH stimulation results in hydrolysis of ovarian phosphoinositides in discrete subcellular compartments while increasing their synthesis at other sites. To investigate this hypothesis, we determined the effects of LH on phosphoinositide metabolism in whole homogenates and mitochondria of ovarian follicles. Medium (3-7 mm) follicles from porcine ovaries were preincubated for 2 h in phosphate (PO4)-free medium with 32PO4, and incubated without or with LH (1 microgram/ml). Phosphatidylinositol (PI) and related compounds, phosphatidic acid (PA), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2), accounted for 40% of the radiolabeled phospholipids in whole homogenates and over 60% in mitochondria from preincubated follicles. After 5 min, LH caused a significant decrease in radiolabeling of PIP2 and PIP in mitochondria, but not in whole homogenates. Luteinizing hormone increased radiolabeling of PIP2, PIP, PI and PA within 10 min in whole homogenates, and within 20 to 30 min in mitochondria. This delayed increase in radiolabeling of mitochondrial phosphoinositides after LH treatment was accompanied by decreases in PIP2, PIP and PI radiolabeling in whole homogenates. Follicles also were preincubated for 4 h with [3H]inositol, then for 15 min with 10 mM LiCl (an inhibitor of inositol phosphate hydrolysis). Inositol phosphate accumulation in 30 min was 2.7 times higher in homogenates of LH-treated follicles then in untreated follicles. Also, LH significantly decreased inositol bisphosphate, but did not change inositol trisphosphate accumulation. Accumulation of inositol phosphates in mitochondria was not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone.

Accumulation of inositol phosphates by granulosa cells from medium follicles of porcine ovaries was studied to determine if hydrolysis of phosphoinositides is stimulated by luteinizing hormone (LH). Although follicle-stimulating hormone (FSH), D-alanine-gonadotropin-releasing hormone (D-ala-GnRH), and dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) had no effect, LH increased accumulation of inositol phosphate (IP), -bisphosphate (IP2), and trisphosphate (IP3) by severalfold. Furthermore, 0.01 microgram LH/ml increased IP3 accumulation threefold, while 0.1 microgram/ml stimulated accumulation of all inositol phosphates. Compared to untreated cells, LH-treated granulosa cells produced approximately twice as much progesterone in 30 min. Preincubation of cells with lithium chloride (LiCl) was necessary to measure IP accumulation, but not IP2 and IP3 accumulations. However, IP2 and IP3 accumulations were higher in LH-treated granulosa after pretreatment with LiCl. Maximal increases in IP3 and IP2 accumulations occurred approximately 15 min and 30 min, respectively, after LH stimulation, whereas the effect of LH on IP accumulation continued for at least 60 min. Granulosa, made permeable to IP3 with saponin treatment, did not hydrolyze [3H]IP3 to [3H]IP2 or [3H]IP. Thus, it is hypothesized that LH stimulates phosphoinositide hydrolysis in granulosa cells, thereby generating putative second messengers.

Animals↗

Characterization and distribution of protein kinase C in ovarian tissue.

Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.

Animals↗

Phosphorylation of mitochondrial proteins in isolated porcine ovarian follicles after treatment with luteinizing hormone.

LH treatment of isolated medium (3- to 7-mm) follicles from porcine ovaries stimulated pregnenolone synthesis by mitochondria isolated from these tissues. This LH stimulation of pregnenolone synthesis was observed regardless of whether exogenous cholesterol was added to the mitochondrial assay system, suggesting that factors besides cholesterol availability may be important for mitochondrial steroidogenesis. Since ovarian mitochondria contain AMP-dependent protein kinase activity, we investigated the role of protein phosphorylation in regulating mitochondrial steroidogenesis. Mitochondria were isolated from different sized follicles and corpora lutea and incubated for 10 min with [gamma-32P]ATP in the absence or presence of 2 microM cAMP. Mitochondrial proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 32P labeling of mitochondrial proteins was determined by autoradiography. In all mitochondrial preparations from the various ovarian tissues, 32P labeling was found only for proteins with apparent mol wt of 44,000 and 55,000. While cAMP addition was necessary for phosphorylation of the 44,000 mol wt mitochondrial protein(s), phosphorylation of the 55,000 mol wt mitochondrial protein(s) occurred independently of cAMP. Intact medium follicles were preincubated for 120 min in medium 199D containing 32Pi, and then incubated in nonradioactive medium without or with LH (1 microgram/ml) for up to 2 h. Mitochondria were isolated from these follicles, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While several mitochondrial proteins were labeled in these experiments, only the 44,000 and 55,000 mol wt proteins were studied. After 30 min of incubation, LH treatment stimulated the phosphorylation of a mitochondrial protein with apparent mol wt of 44,000. However, 32P labeling of this 44,000 mol wt mitochondrial protein decreased in LH-treated follicles after incubation for 60 and 120 min. In untreated follicles, 32P labeling of the 44,000 mol wt mitochondrial protein did not change after 30 min of incubation, but phosphorylation did increase after 60- and 120-min incubations. 32P labeling of the 55,000 mol wt mitochondrial protein increased linearly with time in both untreated and LH-treated follicles. Although cAMP treatment of isolated follicular mitochondria was similar to the LH stimulation of intact follicles with respect to phosphorylation of the 44,000 mol wt mitochondrial protein, cAMP treatment of isolated follicular mitochondria did not stimulate pregnenolone synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Progesterone secretion by granulosa cells from different sized follicles of human ovaries after short term incubation.

Studies on the ability of granulosa to secrete progesterone after hormonal treatment have not addressed the possibility of variations in response associated with the stage of follicular development. Accordingly, granulosa were isolated from follicular fluid obtained from apparently healthy ovaries of patients undergoing tubal bypass. Follicle size was estimated by laparoscopic and ultrasonic measurements, and granulosa cells were prepared by centrifugation and treatment with EGTA and hypertonic sucrose solution to minimize aggregation. Cell viability was determined and 10(6) cells/ml medium 199D were incubated for 24 h without or with ovine LH (1 microgram/ml) or 17 beta-estradiol (5 micrograms/ml). Progesterone secretion by granulosa increased from 6 to 30 ng/24 h/10(6) cells and correlated with follicle sizes of 5--25 mm. Compared to untreated cells obtained from the same follicle, granulosa from follicles ranging from 5--15 mm secreted 2.5 times more progesterone when treated with LH. This effect of LH diminished with further increases in follicle size, with LH having no effect on cells from follicles larger than 20 mm. Compared to untreated cells from the same follicles, 17 beta-estradiol caused a progressive inhibition of progesterone secretion by granulosa that correlated with follicle sizes between 5--18 mm, with almost 60% inhibition occurring in cells from 18-mm follicles. Thereafter, the inhibitory effect of 17 beta-estradiol diminished, and no effect was found in granulosa from follicles larger than 22 mm. These results indicate that LH and 17 beta-estradiol have maximal in vitro effects on progesterone secretion by granulosa from follicles with diameters of 15--18 mm. The change in response to LH and 17 beta-estradiol observed in samples from larger follicles may be due to the fact that these cells already have maximal binding of LH and 17 beta-estradiol to their receptors. Alternatively, it is possible that cells from larger follicles are desensitized to LH and 17 beta-estradiol.

Androstenedione↗

Cyclic AMP-dependent protein kinase in mitochondria and cytosol from different-sized follicles and corpora lutea of porcine ovaries.

cAMP-dependent protein kinase was examined in mitochondria and cytosol prepared from different-sized antral follicles and corpora lutea of porcine ovaries. In all ovarian tissues examined except small follicles, protein kinase-specific activity was significantly higher in mitochondria than in cytosol, with the highest to lowest activities being found in medium (4-6 mm) follicles, large (7-12 mm) follicles, corpora lutea, and small (1-3 mm) follicles, respectively. Using the photoaffinity analogue [32P]8-N3cAMP, two major cAMP binding proteins with Mr = 47,000 (the apparent regulatory subunit of protein kinase Type I) and 54,000-56,000 (Type II) were found in all ovarian preparations. Type II was predominant in the cytosol of all ovarian samples, with the cytosolic Type I to Type II ratio increasing from approximately 0.05 in small and medium follicles top approximately 0.20 in large follicles and corpora lutea. In contrast, ovarian mitochondrial preparations contained relatively more Type I than did cytosol, with the mitochondrial Type I to Type II ratio increasing from approximately 0.50 in small and medium follicles to 0.88 in large follicles and 2.96 in corpora lutea. Also, mitochondrial [4-14C]cholesterol conversion and 3 beta-hydroxysteroid dehydrogenase/isomerase activities increased with follicle size and luteinization. These results suggest that Type I may play a role in the regulation of ovarian mitochondrial steroidogenesis.

Affinity Labels↗

A relationship between adenosine 5'-triphosphate and pregnenolone syntheses by ovarian mitochondria as demonstrated with aminoglutethimide.

Aminoglutethimide, an inhibitor of pregnenolone synthesis, was used to study the relationship between oxidative phosphorylation and steroidogenesis in ovarian mitochondria. In vitro addition of aminoglutethimide to mitochondria prepared from porcine corpora lutea resulted in a decrease in oxygen uptake and an increase in the ADP to oxygen ratio, with maximal changes occurring at a concentration of 300 microM. Similar decreases in oxygen uptake were seen when aminoglutethimide was added to ovarian mitochondria from untreated and PMS gonadotropin-treated immature rats and intact and hypophysectomized mature rats. Also, aminoglutethimide increased the ADP to oxygen ratio in ovarian mitochondria from PMS gonadotropin-treated immature rats and intact mature rats. The addition of aminoglutethimide to luteal mitochondria inhibited their [4-14C]cholesterol conversion activity but increased their ATP synthesis rate by 25%. It is concluded that inhibition of ovarian mitochondrial steroidogenesis by aminoglutethimide is accompanied by both a decrease in oxygen consumption and an increase in ATP synthesis.

Adenosine Triphosphate↗

Increases in mitochondrial steroidogenesis after short term incubation of porcine ovarian follicles with luteinizing hormone.

[4-14C]Cholesterol side chain cleavage, progesterone synthesis, and cytochrome oxidase activities were measured in mitochondria from unincubated and short term incubated large (8-10 mm) follicles isolated from porcine ovaries. Compared to the activity of mitochondria from unincubated follicles, specific [4-14C]cholesterol side chain cleavage activity in mitochondria from follicles incubated with LH (0.05 microgrogram/ml) did not change significantly after 12 h, but increased almost 2-fold after 18 h and 5-fold after 24 h. Also, specific mitochondrial progesterone synthesis activity increased dramatically after incubation of follicles for 24 h with LH. In comparison, mitochondria prepared from follicles incubated without LH showed no significant change in specific [4-14C]cholesterol side chain cleavage or progesterone synthesis activities after 18 h of incubation. While both of these activities increased after incubating follicles 24 h without LH, the values were significantly lower than those observed for preparations from follicles incubated with LH. In contrast to these changes in mitochondrial steroidogenesis, specific cytochrome oxidase activity in mitochondria did not change after incubation of follicles without or with LH. It is concluded that incubation of follicles with LH stimulates the development of mitochondrial steroidogenesis but initially does not affect some components of the respiratory chain.

Animals↗