Incubation In Control Medium Research Articles

Osteoblast-Mediated Resorption of Porous Bioactive SCPC Granules Enhances Bone Regeneration in Human Extraction Sockets

Bone graft materials are widely used in orthopedic and maxillofacial surgeries. The controlled resorbability of the graft material is essential for bone regeneration. Hydroxyapatite and biphasic calcium phosphate bone grafts have poor resorption and limited bone conductive effects. Histology analyses of bone biopsy from SCPC grafted human extraction sockets showed complete bone regeneration and graft resorption in absence of osteoclasts and macrophages. The hypothesis of the present study is that bioactive SCPC inhibits osteoclast’s activity due to the presence of resorbable silica phase in the material. Our objective is to analyze the effect of SCPC dissolution products on the resorption activity of osteoclasts. The conditioned medium was prepared by immersion of SCPC resorbable bioactive SCPC porous granules (Shefabone, Inc, USA) in cell culture medium at various ratios at 37°C for 3 days. The concentration of Si ions released from the SCPC granules into cell culture medium was measured using ICP-OES. Osteoclast precursors derived from human bone marrow were seeded on bone slices and cultured in the conditioned medium containing 10% FBS and osteoclast induction factors. Osteoclast differentiation and resorption were evaluated by TRAP staining and measurement of the volume of resorption pits on the bone slices. Mature multinuclear giant TRAP-positive osteoclasts were observed on the bone substrates after 14 days incubation in control medium containing osteoclast induction factors. In conditioned medium, the number of multinuclear TRAP-positive cells was significantly decreased as the concentration of SCPC dissolved silica increased. The dissolution of silica from SCPC into the culture medium correlates well with down regulation of osteoclast differentiation and the rapid bone regeneration in human bone defects.

Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30μM) for 2–8days; 2) VCD (30μM) for 2days, followed by incubation in control media for 4days (acute VCD exposure); or 3) LY294002 (20μM) for 6days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.

Effects of orexigenic peptides and leptin on melatonin secretion during different photoperiods in seasonal breeding ewes: An in vitro study

The pineal gland (PG) acts as a neuroendocrine transducer of daily and seasonal time through the nocturnal release of melatonin. Here, we examined the interaction of season, orexin, ghrelin, and leptin on melatonin secretion by pineal explants in short-term culture. Glands were collected after sunset from 12 ewes during long days (LD; April and May) and from an additional 12 ewes during short days (SD; October and November). Glands were transected sagittally into strips, with each equilibrated in 2.5 mL of Dulbecco's modified Eagle's medium for 60 min, followed by a 2-h incubation in control medium or medium containing orexin B (10 and 100 ng/mL), ghrelin (10 and 100 ng/mL), or 50 ng/mL of leptin. After a 3-h incubation, some PG explants treated previously with lower doses of orexin or ghrelin were challenged with 50 ng/mL of leptin and those treated with both doses of orexin were challenged with 300 nM of the β-agonist isoproterenol. One milliliter of medium was harvested and replaced from each well every 30 min. Treatment with the low dose of orexin during LD increased melatonin secretion about 110% ( P < 0.01); treatment with a high dose increased melatonin secretion about 47% ( P < 0.001). During the SD period, leptin stimulated ( P < 0.05) melatonin secretion slightly compared with mean melatonin concentration in controls. However, together, orexin and leptin depressed ( P < 0.01) melatonin secretion. Both doses of ghrelin reduced ( P < 0.01) melatonin concentration during the SD season compared with control culture. Addition of ghrelin and leptin to culture medium increased ( P < 0.01) melatonin concentration compared with ghrelin-treated culture and decreased melatonin concentration ( P < 0.01) compared with leptin-treated culture during SD. Isoproterenol stimulated ( P < 0.01) melatonin secretion compared with values observed during the pretreatment period. We conclude that orexigenic peptides (orexin B and ghrelin) and an anorectic peptide (leptin) affect PG directly. The responses of PG to those hormones depend on day length. Moreover, secretion of melatonin from the ovine PG is under an adrenergic regulation.

Synergistic antiproliferative effects of KIT tyrosine kinase inhibitors on neoplastic canine mast cells

Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by (3)H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC(50) values: imatinib: 269 +/- 180 nM, midostaurin: 157 +/- 35 nM, nilotinib: 55 +/- 24 nM, dasatinib: 12 +/- 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC(50) values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors.

Influence of Polychlorinated Biphenyls and Turning Preference on Striatal Dopamine Metabolism

Male BALB/c mice, assessed for spontaneous nocturnal rotation that has been linked with functional differences in striatal dopamine (DA) content, were divided into right (R), left (L), and no turning preference (NP) groups. Both total turning activity and turning in the preferred direction were greatest in the R mice. To determine whether turning preference influences the response to exposure to an environmental toxicant known to reduce striatal DA function, striatal tissues from R, L, or NP mice were exposed to polychlorinated biphenyls (PCBs). In vitro exposure of striata from these mice to varying concentrations of PCBs for 4 h concentration-dependently decreased tissue DA content and increased the concentrations of DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the media, a phenomenon already observed, although of smaller magnitude, with rat tissues. These effects were independent of the turning preference of the mice. Although the DA content initially did not differ among the striatal tissue from R, L, and NP mice, following 4 h of incubation in control medium (without PCBs), there was significantly greater DA content in striata from R mice in comparison to that from either L or NP mice. This difference was also apparent after 6 h of incubation, and it was not due to differences in DA turnover or cytotoxicity. Rather, the greater DA content in the striata of R mice was due to increased DA synthesis, since tyrosine hydroxylase (TH) activity was greater in striata from R mice than in striata from either L or NP mice. These differences in striatal DA synthesis, if present in vivo, may explain the observed greater nocturnal turning activity of the R mice. Additionally, the in vitro analyses suggest that striata from R mice have differential responses to stress compared with striata from L or NP mice. With regard to the effects of PCBs, however, it appears that (1) striata derived from mice with different turning preference are equally sensitive to this dopaminergic toxicant and (2) mice appear more sensitive to PCBs than rats.The project described here was supported in part by grants ES07251 (D.A.L.), ES05879 (N.M.F.), and ES079102 (R.F.S.) from the National Institute of Environmental Health Sciences (NIEHS, NIH), and EPA82581 (R.F.S.) from the U.S. Environmental Protection Agency. The technical assistance of Dr. J. Bemis, K. Brosch, and R. Okoniewski was greatly appreciated.

Effect of homologous follicular fluid from medium-sized and large follicles on in vitro maturation of equine cumulus - oocyte complexes

The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n = 232) and non-compact (n = 183) COCs obtained from a slaughterhouse were incubated separately in the following groups: (1) FF from medium follicles for 24 h; (2) FF from large follicles for 24 h; (3) control (synthetic) medium for 24 h; (4) FF from medium follicles for 24 h then FF from large follicles for an additional 24 h; (5) FF from large follicles for 48 h; and (6) control medium for 48 h. For compact COCs, there was a tendency (P = 0.06) for more COCs incubated in FF from large follicles for 24 h to reach metaphase II compared with those incubated in control medium for 24 h (58% v. 35%, respectively). More (P < 0.05) compact COCs had degenerated after incubation in control medium for 48 h compared with all other groups (51% v. 14-24%, respectively). For non-compact COCs, incubation in FF from medium follicles for 24 h resulted in more (P = 0.05) COCs at metaphase II compared with control medium for 48 h (58% v. 29%, respectively). These results indicate that homologous FF from cyclic mares is a suitable alternative for the IVM of equine COCs and that it may be superior to conventional media for longer (i.e. >24 h) incubation intervals.

Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.

Chronic acidosis-induced alteration in bone bicarbonate and phosphate.

Chronic metabolic acidosis increases urinary calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. In vivo and in vitro studies have shown that metabolic acidosis causes a loss of mineral calcium while buffering the additional hydrogen ions. Previously, we studied changes in femoral, midcortical ion concentrations after 7 days of in vivo metabolic acidosis induced by oral ammonium chloride. We found that, compared with mice drinking only distilled water, ammonium chloride induced a loss of bone sodium and potassium and a depletion of mineral HCO3(-) and phosphate. There is more phosphate than carbonate in neonatal mouse bone. In the present in vitro study, we utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to test the hypothesis that chronic acidosis would decrease bulk (cross-sectional) bone phosphate to a greater extent than HCO3(-) by localizing and comparing changes in bone HCO3(-) and phosphate after chronic incubation of neonatal mouse calvariae in acidic medium. Calvariae were cultured for a total of 51 h in medium acidified by a reduction in HCO3(-) concentration ([HCO(-)]; pH approximately 7.14, [HCO3(-)] approximately 13) or in control medium (pH approximately 7.45, HCO3(-) approximately 26). Compared with incubation in control medium, incubation in acidic medium caused no change in surface total phosphate but a significant fall in cross-sectional phosphate, with respect to the carbon-carbon bond (C2) and the carbon-nitrogen bond (CN). Compared with incubation in control medium, incubation in acidic medium caused no change in surface HCO3(-) but a significant fall in cross-sectional HCO3(-) with respect to C2 and CN. The fall in cross-sectional phosphate was significantly greater than the fall in cross-sectional HCO3(-). The fall in phosphate indicates release of mineral phosphates, and the fall in HCO3(-) indicates release of mineral HCO3(-), both of which would be expected to buffer the additional protons and help restore the pH toward normal. Thus a model of chronic acidosis depletes bulk bone proton buffers, with phosphate depletion exceeding that of HCO3(-).

The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways.

Previous studies have reported a protective role for the von Hippel-Lindau (VHL) gene products against pro-apoptotic cellular stresses, but the mechanisms remain unclear. In this study, we examined the role of VHL in renal cells subjected to chemical hypoxia, using four VHL-negative and two VHL-positive cell lines. VHL-negative renal carcinoma cells underwent apoptosis following chemical hypoxia (short-term glucose deprivation and antimycin treatment), as evidenced by morphologic changes and internucleosomal DNA cleavage. Reintroduction of VHL expression prevented this apoptosis. VHL-negative cells displayed a significant (greater than 5-fold) activation of caspase 9 and release of cytochrome c into the cytosol following chemical hypoxia. In contrast, VHL-positive cells showed minimal caspase 9 activation, and absence of cytochrome c release under the same conditions. Caspase 8 was only minimally activated in both VHL-negative and -positive cells. In addition, VHL-positive cells displayed a striking up-regulation of Bcl-2 expression (5-fold) following chemical hypoxia. Antisense oligonucleotides to Bcl-2 significantly down-regulated Bcl-2 protein expression in VHL-positive cells and rendered them sensitive to apoptosis. Overexpression of Bcl-2 in VHL-negative cells conferred resistance to apoptosis. Our results suggest that VHL protects renal cells from apoptosis via Bcl-2-dependent pathways.

Influence of co-culture with established human endometrial epithelial and stromal cell lines on sperm movement characteristics.

The effects of co-culture of human spermatozoa with human immortalized endometrial cells - epithelial or stromal - on sperm movement characteristics, including hyperactivation, were studied using computer-assisted sperm analysis (CASA). Epithelial and stromal cell types could be separated following 8-10 days of culture of endometrial cells originating from human biopsies. Both cell types were immortalized by the SV 40 large T antigen. Co-incubation of sperm with epithelial and stromal monolayers enhanced the rate of hyperactivation: 24.9% (P <0.05) and 17.8% (P = 0.05) versus 9.5% as control, respectively, whereas the majority of motility parameters remained unchanged. Conditioned media had no effect upon sperm parameters, including hyperactivation. Co-incubation with either monolayer was able to maintain sperm motility over a longer period than incubation in control medium alone. In four patients whose spermatozoa did not exhibit hyperactivation, co-incubation with epithelial cells, but not conditioned medium, allowed normal rates of hyperactivation (range: 6.9-15.6%).

Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa.

Our laboratory has recently shown that in vitro-cultured oviductal cells secrete sperm motility maintaining factor(s). Since the binding of oviductal proteins to spermatozoa (SPZ) has been demonstrated in many species, the motility factor was postulated to bind the membranes of SPZ. Therefore, the current study was performed to evaluate which proteins from in vivo oviductal secretions bind to sperm membranes, to characterize binding conditions, and to evaluate the effect of this binding on sperm survival. Bovine oviducts were dissected, and oviductal cells and fluid were collected by pressing the oviductal tube with a glass slide. This mixture was incubated in Tris-EDTA buffer at 37 degrees C for 30 min, and the cells were washed twice by centrifugation. The supernatant containing oviductal fluid proteins (OFP) was reserved, filtered, frozen (for later motility tests), or lyophilized and labeled with 125I. Frozen-thawed SPZ were incubated either immediately, following capacitation, ionophore-induced acrosome reaction, death by heating, or flagellar removal with labeled OFP for 30 min. The resulting pellet after three washes was dissolved in SDS and submitted to 10% SDS-PAGE. An autoradiogram showed that 72, 66, 39, 38, and 36 kDa proteins bind strongly to the five types of SPZ used, and that this binding is very specific, since unlabeled OFP inhibited binding while serum proteins did not. Furthermore, for 39, 38, and 36 kDa proteins, the presence of calcium in the incubation medium was essential for dose-dependent binding, whereas magnesium was not. Preincubation of SPZ for 30 min at 37 degrees C with oviductal fluid, followed by one wash and 6 hr of incubation in control media, showed that the percentage of motile SPZ is significantly higher (52 +/- 6%) compared with SPZ not preincubated with oviductal fluid (24 +/- 6%; P < 0.01). In summary, a limited number of proteins from oviductal secretions bind to the surface of bovine SPZ only in the presence of calcium, and this binding appears to be important for subsequent sperm viability.

Interleukin-12 synergizes with interleukin-2 to generate lymphokine-activated killer activity in peripheral blood mononuclear cells cultured in ovarian cancer ascitic fluid

The ascites-associated lymphocytes in ovarian cancer have altered immunologic function, and cell-free ascitic fluid has immunomodulating properties. We determined (1) whether interleukin (IL)-2 could induce lymphokine-activated killer (LAK) activity in normal peripheral blood mononuclear cells (PBMC) cultured in ovarian cancer ascitic fluid, and (2) whether IL-12 could synergize with IL-2 to generate LAK activity in normal PBMC cultured in ascitic fluid. Normal PBMC were cultured in control medium and in media consisting of 50% ascitic fluid (ascitic medium), with and without IL-2 and IL-12. Cell activation to assess LAK activity (cell lysis) was determined in a 51Cr-release assay with the tumor cell lines FMEX and SKOV3 as target cells. To determine a possible mechanism for any synergistic effect, the expression of perforin, a pore-forming protein, was determined by Northern blot analysis. Interleukin-2 alone could not induce LAK activity in normal PBMC cultured in 50% ascitic fluid for up to 3 days. Interleukin-12 did mediate some or minimal LAK activity after 1, 2, or 3 days of incubation in control medium or in 50% ascitic fluid. When IL-2 and IL-12 were used in combination, PBMC cultured for 3 days in 50% ascitic fluid had remarkably high lytic activity against FMEX and SKOV3 tumor cells. In some experiments, this cytotoxicity was greater than that in PBMC cultured in control medium with IL-2 and IL-12. Lower concentrations of IL-12 (1 U/mL) with IL-2 (100 U/mL) were as effective as, and often more effective than, higher doses of IL-12 with IL-2. Very low-dose IL-12 (0.01-0.03 U/mL) in combination with IL-2 also induced a range of cytotoxicities. Only the combination of IL-2 and IL-12 up-regulated expression of perforin mRNA in ascitic medium. The cytotoxicity responses of PBMC cultured in ascitic fluid in the presence of IL-2 and IL-12 are complex. Low-dose IL-2 and IL-12 can overcome the inhibitory property of ascitic fluid on LAK generation and can restore and enhance cytotoxic activity, possibly by reconstituting the expression of perforin. These findings may have therapeutic potential.

Effect of Hyaluronic Acid on Rabbit Profundus Flexor Tendon Healing in Vitro

We performed an in-depth biomechanical evaluation of the effect of hyaluronic acid (HA) on the healing of rabbit profundus tendons cultured in vitro. Seventy-eight flexor tendons from 13 rabbits were transected and reapproximated at their Zone II midpoints. Tendons were divided into left and right forepaw groups. Each tendon from the left forepaw group was incubated in one of four possible culture media: control (no HA), low (0.1 mg/ml), medium (0.5 mg/ml), or high (1.0 mg/ml) HA media. HA was added on the first day of incubation. Each tendon from the right forepaw group was cultured in low, medium, or high concentrations of HA, but HA was added after 1 week of incubation in control media. All tendons were cultured for 8 weeks, after which time tenorrhaphies were disrupted and the following biomechanical parameters were determined: apparent maximum stress, apparent strain at apparent maximum stress, normalized energy absorption, and tangent modulus before failure. Comparisons using these parameters showed no statistically significant differences among the various tendon groups. We believe this is the first study of its kind to show no effect of hyaluronic acid on the functional strength of tendon after healing in vitro.

Effects of pentoxifylline on sperm motility and hyperactivation in normozoospermic and normokinetic semen

To determine the effects of in vitro incubation with pentoxifylline on sperm motion characteristics of spermatozoa from normozoospermic, normokinetic specimens. Prospective, controlled experiment. Andrology laboratory, university-based fertility center. Healthy, untreated male partners of couples attending the fertility center. Each specimen was washed, pelleted by centrifugation, then resuspended in human tubal fluid medium (HTF). Two portions were incubated at 37 degrees C, one with pentoxifylline (final concentration = 1 mg/mL = 3.6 mM) and the other without pentoxifylline (control). After 1 hour, the pentoxifylline-treated portion was divided: one half was washed to remove pentoxifylline, then further incubated in HTF; the other half remained incubated in HTF with pentoxifylline. Motility, hyperactivation, amplitude of lateral head displacement (ALH), curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), beat-cross frequency. Incubation with pentoxifylline did not increase motility, VSL, LIN, or beat-cross frequency but did significantly increase HA, VCL, and ALH at 1, 2, and 4 hours, compared with control. Incubation in control medium without pentoxifylline did not significantly increase any of the parameters measured. After 24 hours of incubation with or without pentoxifylline, all parameters measured were significantly decreased, with the exception of LIN. Pentoxifylline does not increase percentage motility of washed spermatozoa in capacitation medium. Enhancement of sperm HA by pentoxifylline in capacitation medium occurs with normozoospermic, normokinetic semen specimens. This effect persists for up to 4 hours when pentoxifylline is removed from the medium after 1 hour of incubation.

Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10 −6, 10 −4 and 10 −2 M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10 −2 M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10 −2 M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10 −2 M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.

Phosphorus and proton magnetic resonance spectroscopic studies on the relationship between transparency and glucose metabolism in the rabbit lens

We report the results of a series of nuclear magnetic resonance (NMR) experiments designed to investigate the relationship between particular aspects of glucose metabolism and cataract formation in the rabbit lens. The glucose metabolism of the rabbit lens incubated in TC-199 medium containing 5·5 m m glucose, in glucose-deficient medium, and in modified Earle's medium containing 5·5 m m glucose devoid of NaCl, is examined in conjunction with the assessment of lens transparency. Significant age-dependent differences in the phosphorus metabolite profile and in hexosemonophosphate shunt flux, as measured by NMR, were observed in lenses incubated in TC-199 medium containing 5·5 m m glucose. Incubation in glucose-deficient medium for 8 hr results in significant increases in the levels of inorganic phosphate and phosphomonoesters, and decreases in ATP and l-α-glycerolphosphate. These levels regain near-normal values after 24 hr incubation in control medium containing 5·5 m m glucose. By contrast, shunt flux is three times the basal level during the recovery period. Lens clarity, as assessed by slit lamp micrography, was maintained throughout the duration of the experiment. Incubation of adult and juvenile lenses for 18 hr in Earle's medium (pH 7·4 or 9·2) containing 5·5 m m glucose, and no NaCl, results in uniform lenticular opacification within 18 hr and changes in ultrastructure of the epithelial and cortical lens fiber cells. No statistically significant change in the NMR visible phosphorus metabolite profile or intralenticular pH is observed for the adult rabbit lens relative to a lens incubated under control conditions. For the juvenile rabbit lens, small, but statistically significant differences in the levels of dinucleotide and uridinediphosphoglucose were observed. Shunt flux, in contrast, is increased two-fold. These results demonstrate that the NMR visible phosphorus metabolite profile of the lens does not necessarily correlate with transparency, and that hexosemonophosphate shunt activity provides a sensitive measure of prior or current lenticular stress.

Effects of water acidification on the decomposition of Juncus bulbosus L.

Several field and laboratory experiments were performed to study the impact of acidification on the decomposition of Juncus bulbosus L. The breakdown of coarse detritus of this aquatic macrophyte appeared to be very slow. After 7 weeks of incubation in an acidified and a circumneutral moorland pool the remaining fraction of Juncus leaves varied between 46 and 75% of the original organic weight. Differences in decay patterns were observed among both systems. Furthermore, the breakdown rates were highly dependent on the season. Highest weight loss rates were recorded at high pH during the summer period. In acidified water and during the winter period the breakdown rates were reduced. Nutrient dynamics of detritus in the circumneutral soft water and the acidified system differed remarkably. In litter bags laid down on the bottom surface of the acidified moorland pool the density and diversity of macroinvertebrate assemblages was generally lower than in the bags immersed in the non-acidified body of water. In particular, the abundance of detritivores and herbivores at low pH was strongly reduced. However, in winter qualitative and quantitative differences in the macrofauna in the litter bags did not significantly affect decay rates of detritus. Laboratory studies revealed that the pH, bicarbonate concentration and aluminium content of the water particularly influenced the rate of decomposition of the pre-dried Juncus material. The extent of leaching and autolysis and the relative importance of fungi and bacteria in the decomposition of fresh Juncus leaves were studied using antifungal and antibacterial agents. In the laboratory leaching and autolysis accounted for 28–32% of the organic weight loss of coarse detritus. Leaching and autolysis caused the highest weight loss in the strongly acid medium. After 37 days of incubation in control media with pH 3.5 and 5.6 total organic weight loss was 55 and 59%, respectively. At pH 3.5 and 5.6 microbial decomposition accounted for organic weight losses of 23 and 31%, respectively. At low pH microbial decomposition was dominated by fungi, whereas, at pH 5.6 both fungi and bacteria played an important role in the breakdown processes. The contribution of macroinvertebrates to the breakdown of Juncus detritus seemed to be of minor importance.

Structure-activity relationship of retinoids on lobuloalveolar differentiation of cultured mouse mammary glands.

The influence of 28 retinoids on the structural differentiation of mouse mammary glands in whole organ cultures was examined. Mammary glands from BALB/c mice pretreated with steroids (estradiol and progesterone) and cultured in the presence of lactogenic hormones (insulin, prolactin, hydrocortisone and aldosterone) differentiate into lobuloalveolar structures. These differentiation processes were inhibited by retinoic acid at 10(-6) M and by the arotinoid containing tetramethylated tetraline (Ro 13-7410) at 10(-10) M. The presence of this arotinoid for as little as 5 min, followed by incubation in control medium, irreversibly suppressed lobuloalveolar differentiation. After pre-incubation of mammary glands in control medium for 3 days, the arotinoids no longer suppressed the differentiation of lobuloalveolar structures. Structure-activity relationships of selected retinoids with marked differences in their biological activity were similar to those reported. The mouse mammary gland in whole organ culture may be a suitable and useful bioassay system for selecting retinoids which affect differentiation processes and may thus be helpful in the treatment of oncological diseases.

Rapid depletion of somatostatin in isolated mouse pancreatic islets after treatment with cysteamine.

Cysteamine (CSH; beta-mercaptoethylamine) is known to deplete pancreatic somatostatin without affecting the insulin or glucagon content. It may therefore be useful for studies of intra-islet regulation of hormone release. In the present study injection of CSH (60 mg/kg body weight) to mice decreased the somatostatin content of their isolated pancreatic islets to 50% in 1 h and 30% in 4 h as compared to islets of non-injected controls. Exposure of isolated mouse islets to CSH (100 micrograms/ml) for either 0.5 h followed by incubation in control medium for 3.5 h, or continuously for 4 h, decreased the somatostatin content to about 40% of the controls. There was no change in the islet content of insulin or glucagon. Islets pretreated with CSH (100 micrograms/ml) for 1 h in vitro showed a decreased glucose stimulation of both oxygen consumption and glucose oxidation. Measurements of insulin release after a similar preincubation of the islets indicated an increased basal release and an attenuated glucose stimulation. It is concluded that CSH rapidly decreases islet somatostatin both in vivo and in vitro. This depletion may lead to a loss of tonic inhibition by islet somatostatin on basal insulin release. It is, however, more plausible that the increased basal insulin release reflected a direct effect of CSH on the islet beta-cells.

Ionic requirements for in vitro retrograde axonal transport of acetylcholinesterase

The ionic requirements for retrograde axonal transport of acetylcholinesterase were studied in vitro in desheathed spinal nerves of the bullfrog; the accumulation of enzyme activity distal to a ligature served to evaluate the status of retrograde transport. After an 18 h incubation in control medium, the 2 mm segment of nerve immediately distal to the ligature contained approximately twice as much acetylcholinesterase activity as other more distal segments. Accumulation of acetylcholinesterase distal to the ligature was reduced during incubation in Ca 2+-free medium plus 1 mM EGTA; retrograde transport was not diminished by the substitution of sucrose for the NaCl of the medium. In comparison with ionic requirements for orthograde fast axonal transport of protein, retrograde transport of acetylcholinesterase thus appears to share a Ca 2+ requirement, but not a requirement for NaCl.