The interaction of genes and environment on percent of juniper in the diet of goats divergently selected for high or low juniper consumption.

Diet selection and preference by grazing animals are determined by genetic and environmental factors that interact and affect their efficacy for managing vegetation as targeted grazers and developing animals adapted to local grazing environments. The effect of the rearing environments on the consumption of juniper (Juniperus spp.) by goats that for 15 years were divergently selected for high (J+) or low (J-) percent juniper in their diet was investigated. To test the effect of rearing environment, at the end of the breeding season, pregnant does from both selection lines were grazed on either juniper-infested (JIR) or juniper-free (JFR) rangelands until their kids were weaned at about 75 days of age. Fecal samples were analyzed with fecal near-IR spectroscopy to determine the percent juniper in the diet. Fecal samples were collected from does on JIR when their offspring were 30 days of age and at weaning. Then, does that raised kids in both rearing environments grazed a common JIR pasture for a 28-day adaptation period before collecting fecal samples. After weaning, kids from both rearing environments grazed JIR for 22 days before collecting fecal samples. The J+ does always consumed more (P < 0.001) juniper than J- does, demonstrating different maternal role models for kids reared in the JIR environment. There was no effect of rearing environment (P = 0.488) or rearing environment × selection line interaction (P = 0.096) when J- and J+ does grazed a common JIR pasture. The percentage of juniper in J- kid diets (7%) was the same regardless of the rearing environment. However, the rearing environment did affect the percentage of juniper in the diet of J+ kids, resulting in a gene-environment interaction (P = 0.022). The percentage of juniper in the diet of J+ kids reared in JFR (16%) and JIR (24%) were about two and three times higher than J- kids, respectively, indicating that genetics and the rearing environment contributed about equally to the increase in the percentage of juniper in the J+ kid diets. Regardless of the rearing environment, the J+ kids had a higher percentage of juniper in their diets than J- kids (P < 0.001). Compared to males, female kids had a higher percentage of juniper in their diets (12 vs 17%, respectively; P = 0.002). The ability to select animals with specific dietary preferences holds promise for targeted grazing strategies to restore degraded rangelands, with potential applications in conservation and ecosystem management.

Effects of feeding ground redberry juniper (Juniperus pinchotii) to gestating ewes on pre- and postpartum performance, serum metabolites and hormones, milk fatty acid composition, and progeny preweaning performance.

The objective of this research was to evaluate effects of replacing sorghum × Sudangrass hay with ground juniper in gestating ewe supplements on pre- and postpartum growth performance, serum metabolites and hormonal concentrations, milk fatty acid composition, and progeny preweaning performance. In a completely randomized design, commercial Rambouillet ewes (age = 3 to 5 yr; initial BW = 65.2 ± 1.6 kg) on a base diet of long-stem sorghum × Sudangrass hay were assigned to 1 of 4 dietary supplements in which ground juniper replaced 0% (CNTL), 33% (18JUN), 66% (36JUN), or 100% (54JUN) of the ground sorghum × Sudangrass hay in a pelleted supplement with ground juniper from d 38 ± 4 of gestation to 2 d postpartum. Treatment DM diet intake overall (g/kg BW) in ewes receiving no juniper was similar ( ≥ 0.38) to that of those receiving increasing concentrations of juniper. Changes in ewe BW and BCS were similar ( ≥ 0.24) in ewes throughout gestation. All serum metabolites and hormones were within normal clinical ranges; however, serum IGF-1 decreased linearly ( = 0.003), alanine increased (linear; = 0.003), and serum Na decreased (linear; = 0.049) as the percentage of juniper increased in the diet. Ewe milk fatty acid composition was similar ( > 0.05) for the majority of fatty acids across treatment groups, with the exception of arachidonic acid (C20:4n6) being greater ( < 0.02) in 54JUN vs. CNTL ewe milk. Lamb birth weights were similar ( = 0.13), whereas lamb ADG tended to differ (quadratic; = 0.06) from d 0 to 14, with 18JUN being the least. At weaning, BW tended ( = 0.09) to linearly decrease in lambs born to ewes consuming greater concentrations of juniper but were not different ( = 0.26) between CNTL and 18JUN, 36JUN, and 54JUN. Results indicated that feeding increasing levels of ground juniper in supplements did not negatively alter ewe performance or serum metabolites and hormones measured pre- and postpartum. Lamb birth weight and preweaning performance appeared unaffected by maternal consumption of ground juniper containing supplements. Results also provide novel information regarding the effects of plant secondary compound consumption throughout pregnancy on ewe and progeny performance and health.

Effects of Juniperus species and stage of maturity on nutritional, in vitro digestibility, and plant secondary compound characteristics.

Rising feed costs and recurring feed shortages necessitate the investigation into alternative and underutilized feed resources. Nutritional characteristics of species are either unknown or limited to leaves and ground material from small stems. Therefore, the objective was to quantify nutritional characteristics, 48-h true IVDMD (tIVDMD), microbial gas production, and secondary compound characteristics of entire woody plant material of 4 species-, , , and -at immature and mature stages of growth. Immature plants had greater CP concentrations and lower NDF concentrations ( < 0.001) than mature plants regardless of species. Mature plants also had greater ( < 0.001) concentrations of ADF compared with immature plants with the exception of . In general, immature , , and had greater ( < 0.02) tIVDMD and total 48-h and asymptotic gas production than mature plants. Immature and plants were more digested (tIVDMD; < 0.001) than immature and , but tIVDMD did not differ in mature plant material across species. Condensed tannins (CT) were greater ( < 0.001) in immature and than mature plants; differences in CT concentrations among immature species were also detected ( < 0.04). Volatile oil yields were similar across maturity and species with 1 exception: immature yielded more ( < 0.02) volatile oil than mature material. Volatile oil composition across species varied and contained a range of 65 to 70 terpene compounds. The dominant terpenes across species were generally greater ( < 0.05) in immature vs. mature plant material with the exception of . Labdane acids were negligible in , , and and greater in ( < 0.001). Ground material from mature juniper species, although inferior in nutritional quality compared with immature plants, is comparable to traditional low-quality roughage ingredients. Given that has been successfully fed in lamb feedlot diets, the similarities of , and suggest that all three species have potential to be effective roughage ingredients.

Pharmacokinetic differences in exposure to camphor after intraruminal dosing in selectively bred lines of goats.

A pharmacokinetic dosing study with camphor was used to determine whether selection lines of high-juniper-consuming goats (HJC, n = 12) and low-juniper-consuming goats (LJC, n = 12) differed in their respective disposition kinetics. Postdosing plasma camphor concentrations were used to examine whether a timed single blood sample collected after intraruminal administration of camphor would be a useful screening test to aid in the identification of HJC. Yearling female Boer x Spanish goats (n = 24) received a single intraruminal dose of monoterpene cocktail (0.270 g/kg of BW) containing 4 different monoterpenes that represented their composition previously reported for Ashe juniper (Juniperus ashei). Camphor, the predominant monoterpene in Ashe juniper, was 49.6% of the mix and was the monoterpene analyzed for this study. Blood samples were taken at 15 time points from 0 to 8 h after dosing. Concentrations of camphor were measured in plasma using solid phase extraction and gas chromatography/flame-ionization detection analysis. Maximal plasma concentration of camphor was greater for LJC than HJC (P = 0.01), and area under the curve extrapolated to infinity was greater for LJC than HJC (P < 0.01). Total systemic exposure (area under the curve) to camphor was 5 times less in HJC goats. We conclude that 1) HJC goats possess internal mechanisms to reduce the bioavailability of camphor, and 2) a blood sample taken at 45 min or at 60 min after intraruminal administration of camphor may be useful for identifying HJC individual animals from within large populations of goats.

Heritability of juniper consumption in goats.

Data from goats (n = 505), collected over a 4-yr period, were used to estimate the heritability of juniper consumption. Juniper consumption was determined by near-infrared spectroscopy on fecal samples (n = 1,080) collected from female Boer-cross goats grazing pastures with a variety of plants, including juniper. The animals with records were progeny of 72 sires. Individual goats had from 1 to 4 observations over a 4-yr period. Predicted juniper consumption for individual observations ranged from -5 to +62% of the diet. Data were analyzed with a mixed model that included management group as a fixed effect, BW as a covariate, and permanent environment, animal, and residual as random effects. Management group was a significant source of variation. Least squares means of juniper consumption, as a percentage of the total intake, for management groups varied from 19 to 47%. Heritability of juniper consumption was 13%. Repeatability of juniper consumption was 31%. These results suggest that progress to selection for goats that will consume greater amounts of juniper is obtainable, but is expected to be slow.

Protein scaffolds, lipid domains and substrate recognition in protein kinase C function: implications for rational drug design.

Protein kinase C (PKC) represents a family of lipid-regulated protein kinases with ubiquitous expression throughout the animal kingdom. High fidelity in PKC phosphorylation of intended target substrates is crucial for normal cell and tissue function. Therefore, it is likely that multiple interdependent factors contribute to determining substrate specificity in vivo, including divalent cation binding, substrate recognition motifs, local lipid heterogeneity and protein scaffolds. This review provides an overview of targeting mechanisms for the three subclasses of PKC isoforms, conventional, novel and atypical, with an emphasis on how they bind to substrates, lipids/lipid microdomains and multifunctional protein scaffolds. The diversity of interactions between PKC isoforms and their immediate environment is extensive, suggesting that systems biology approaches including proteomics and network modeling may be important strategies for rational drug design in the future.

A fecal near-infrared reflectance spectroscopy-aided methodology to determine goat dietary composition in a Mediterranean shrubland.

An ecologically sound approach to the problem of brush encroachment onto Israeli rangeland might be their utilization by goats, but better knowledge of the feeding selectivity and ability of goats to thrive in encroached areas is required to devise viable production systems. Direct observation of bites could provide precise and accurate estimates of diet selection, but construction of a sufficiently large database would require too much time. The present study describes the first attempt to construct fecal near-infrared reflectance spectroscopy (NIRS) calibrations of the botanical and nutritional composition of the diet, and of the total intake of free-ranging goats, based on reference values determined with bite-count procedures. Calibration of fecal NIRS was based on 43 observations encompassing 3 goat breeds and 4 periods (spring, summer, and fall of 2004, and spring of 2005). Each observation comprised 242 min of continuous recording of the species and bite-type category selected by a single animal, on each of 2 consecutive days. The mass and chemical quality of each species and bite-type category-a total of more than 200,000 bites-were determined by using the simulated bite technique. Associated feces were scanned in the 1,100- to 2,500-nm range with a reflectance monochromator. Fecal NIRS calibrations had reasonable precision for dietary percentages of the 3 main botanical components: herbaceous vegetation (as one category; R(2) = 0.85), Phillyrea latifolia (R(2) = 0.89), and tannin-rich Pistacia lentiscus (R(2) = 0.77), with SE of cross-validation (SECV) of 7.8, 6.3, and 5.6% of DM, respectively. The R(2) values for dietary percentages of CP, NDF, IVDMD, and polyethylene glycol-binding tannins were 0.93, 0.88, 0.91, and 0.74, respectively, with SECV values of 0.9, 2.1, 4.3, and 0.9% of DM, respectively. The R(2) values for intakes of herbaceous vegetation, P. latifolia, and P. lentiscus were 0.80, 0.75, and 0.65, with SECV values of 71, 64, and 46 g of DM/d, respectively. The R(2) values for the daily nutrient intakes were below 0.60. Fecal NIRS data can be used to expand the databases of botanical and nutritional dietary composition when observed and resident animals graze simultaneously, but intakes should be calculated from fecal NIRS-predicted dietary DM composition and an independent evaluation of DMI.

Effects of breed, sex, and age on the variation and ability of fecal near-infrared reflectance spectra to predict the composition of goat diets.

The effects of breed, sex, and age of goats on fecal near-infrared reflectance spectroscopy-predicted percentage juniper in the diet were investigated, as were spectral differences in feces from goats differing in estimated genetic merit for juniper consumption. Eleven goats from each breed, sex, and age combination, representing 2 breeds (Angora and meat-type), 3 sex classifications (female, intact male, and castrated male), and 2 age categories [adult and kid (less than 12 mo of age)] were fed complete, pelleted rations containing 0 or 14% juniper. After 7 d on the same diet, fecal samples were collected for 3 d, and the spectra from the 3 replicate samples were averaged. Fecal samples were assigned to calibration or validation data sets. In a second experiment, Angora and meat goats with high or low estimated genetic merit for juniper consumption were fed the same diet to determine the effect of consumer group on fecal spectra. Feces were scanned in the 1,100- to 2,500-nm range with a scanning reflectance monochromator. Fecal spectra were analyzed for the difference in spectral characteristics and for differences in predicted juniper in the diet using internal and independent calibration equations. Internal calibration had a high precision (R(2) = 0.94), but the precision of independent validations (r(2) = 0.56) was low. Spectral differences were affected by diet, sex, breed, and age (P < 0.04). However, diet was the largest source of variation in spectral differences. Predicted percentage of juniper in the diet also showed that diet was the largest source of variation, accounting for 95% of the variation in predictions from internal calibrations and 51% of the variation in independent validations. Predictions from independent calibrations readily detected differences (P < 0.001) in the percentage of juniper in the 2 diets, and the predicted differences were similar to the actual differences. Predicted juniper in the diet was also affected by sex. Feces from goats from different juniper consumer groups fed a common diet were spectrally different, and the difference may have resulted from a greater intake by high- compared with low-juniper-consuming goats. Fecal near-infrared reflectance spectroscopy predictions of botanical composition of diets should be considered an interval scale of measurement.

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.

Reproductive performance of ewe lambs from ewes from different selection practices with or without induced estrus.

Three groups of ewe lambs born in May (experiment 1; n=211) or April (experiment 2; n=174) were used to evaluate the effects of selection line and induction of estrus on pregnancy rate. Experiment 1 was a single factor experiment with induction of estrus as the main effect. In early December, May-born Targhee (n=82) and Rambouillet x Targhee (n=129) ewes were randomly assigned within body weight to one of two treatment groups: control or induction of estrus. Experiment 2 was designed in a 2x2 factorial array with the main effects of induction of estrus or selection line. In early November, April-born Targhee lambs (n=174) from two distinct selection lines were either treated as controls or received an estrus induction treatment. The two lines included an unselected control line of randomly bred ewes and a line that had been selected since 1976, based on the weight of lamb weaned. Ewes from each line were randomly assigned within body weight to one of the treatment groups. In experiments 1 and 2, estrus was induced using MAP pessaries. Pessaries were inserted for 12 days. At the time of pessary removal, ewe lambs received 400 IU eCG i.m. All ewe lambs were bred in multi-sire pens. Pregnancy rate and fetal numbers were determined either by lambing data or real-time ultrasound. Body weight, lambing date and fetal numbers were analyzed by GLM, and remaining variables were analyzed by CATMOD. For experiment 1, estrus induction increased (P<0.01) pregnancy rates (61 versus 31%) and number of fetuses estimated by real-time ultrasound (79 versus 35%) compared to control ewe lambs. Pregnancy rate and fetal number were increased (P<0.01) for the 1st year compared to the 2nd year. For experiment 2, estrus induction tended to increase (P<0.07) pregnancy rate, and pregnancy rate differed (P<0.01) between selection lines. Estrus induction increased (P<0.05) fetal numbers (0.96) compared to controls (0.77). Fetal numbers were greater (P<0.01) for the selected line (1.06) compared to random bred controls (0.67). Average date of lambing was earlier in both experiments for the estrus-induced ewe lambs compared to controls. These results indicate that induction of estrus can be recommended if increased reproduction is desired for ewe lambs.

Deep venous thrombosis and pulmonary embolus after face lift: a study of incidence and prophylaxis.

Deep venous thrombosis and pulmonary embolus are known risks of surgery. However, the incidence of these conditions in face lift is unknown. In this study, the incidence of deep venous thrombosis/pulmonary embolus after face lift is studied and factors associated with thromboembolic complications are evaluated. One-third of the active members of the American Society for Aesthetic Plastic Surgery were randomly selected. Participating surgeons completed a one-page survey providing information on face-lift procedures during a 12-month study period. A response rate of 80 percent was achieved, with 273 of the 342 surgeons responding to the survey. A total of 9937 face-lift procedures were reported in the 1-year study period. There were 35 patients with deep venous thrombosis (0.35 percent), 14 patients with pulmonary embolus (0.14 percent), and 1 patient death in the series. Although 43.5 percent of patients underwent face lift under general anesthesia, 83.7 percent of deep venous thrombosis/pulmonary embolus events occurred with general anesthesia. For prophylaxis for deep venous thrombosis/pulmonary embolus, 19.7 percent of the surgeons used intermittent compression devices, 19.6 percent used thromboembolic disease hose or Ace wraps, and 60.7 percent used no prophylaxis. Of patients developing deep venous thrombosis/pulmonary embolus, 4.1 percent were treated prophylactically with intermittent compression devices, 36.7 percent with thromboembolic disease hose/Ace wraps, and 59.2 percent with no prophylaxis. It was found that deep venous thrombosis/pulmonary embolus after face lift is a measurable complication experienced by one of nine surgeons surveyed. Deep venous thrombosis/pulmonary embolus is more likely to occur when the procedure is performed under general anesthesia. The majority of plastic surgeons surveyed used no prophylaxis for deep venous thrombosis when performing face-lift procedures. Intermittent compression devices were associated with significantly fewer thromboembolic complications, whereas Ace wrap/thromboembolic disease hose afforded no protection against deep venous thrombosis/pulmonary embolus when used alone. In conclusion, aesthetic surgeons should consider adopting intermittent compression devices when performing face lift under general anesthesia.

Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes.

Protein kinase C-epsilon (PKC-epsilon) plays a central role in cardiac cell signaling, but mechanisms of translocation and anchoring upon activation are poorly understood. Conventional PKC isoforms rely on a rapid Ca2+-mediated recruitment to cell membranes, but this mechanism cannot be employed by PKC-epsilon or other PKC isoforms lacking a Ca2+-binding domain. In this study, we used recombinant green fluorescent protein (GFP) fusion constructs and confocal microscopy to examine the localization, kinetics, and reversibility of PKC-epsilon anchoring in permeabilized rat cardiac myocytes. PKC-epsilon-GFP bound with a striated pattern that co-localized with alpha-actinin, a marker of the Z-line of the sarcomere. Binding required activation of PKC and occurred slowly but reversibly with apparent rate constants of k(on) = 4.6 +/- 1.2 x 10(3) M(-1) x s(-1) and k(off) = 1.4 +/- 0.5 x 10(-3) s(-1) (t1/2 = 8 min) as determined by fluorescence recovery after photobleaching and by perfusion experiments. A truncated construct composed of the N-terminal 144-amino-acid variable region of PKC-epsilon (epsilonV1-GFP), but not an analogous N-terminal domain of PKC-delta, mimicked the Z-line decoration and slow binding rate of the full-length enzyme. These findings suggest that the epsilonV1 domain is important in determining PKC-epsilon localization and translocation kinetics in cardiac muscle. Moreover, PKC-epsilon translocation is not a diffusion-controlled binding process but instead may be limited by intramolecular conformational changes within the V1 domain. The k(off) for epsilonV1-GFP was two- to threefold faster than for full-length enzyme, indicating that other domains in PKC-epsilon contribute to anchoring by prolonging the bound state.

Genetic and phenotypic parameters for dietary selection of mountain big sagebrush (Artemisia tridentata Nutt. ssp. vaseyana.

The heritability of diet selection for mountain big sagebrush (Artemisia tridentata Nutt. ssp. vaseyana [Rydb] Beetle) by grazing sheep was estimated from fecal samples collected from 549 Rambouillet ewes. Fecal samples were collected in September and October during 1996 and 1997 from free-grazing ewes on intermountain sagebrush-bunchgrass rangelands at the U.S. Sheep Experiment Station in Idaho. The total number of fecal samples was 1,949. Fecal samples were evaluated for composition of big sagebrush by near-infrared spectroscopy. Percentage of sagebrush in the diet was less in September than in October (21.6 vs 31.7%, respectively). Single-trait and bivariate derivative-free REML analyses were performed to genetically compare percentage of sagebrush in the diet in September and October. Heritability estimates were similar between September and October measurements (0.25 and 0.28, respectively). The genetic correlation between September and October percentages of sagebrush in the diet was high (0.91), implying that there is strong genetic similarity between September and October measurements and that an annual measurement may be sufficient for selection. These results contribute to a greater understanding of dietary preferences in freely grazing sheep, and suggest opportunities to improve production efficiency and forage management through selection for dietary preferences.

Thyroid hormone regulates slow skeletal troponin I gene inactivation in cardiac troponin I null mouse hearts.

Two main troponin I genes, cardiac (cTnI) and slow skeletal (ssTnI), are expressed in the mammalian heart under the control of a developmentally regulated program. ssTnI is expressed first in embryonic and fetal heart, and is then downregulated by an unknown mechanism after birth. Unlike other contractile protein genes, ssTnI is not re-expressed during hypertrophy or end-stage heart failure in rats and humans. In the present study, we also show that ssTnI re-expression does not occur in hypertrophic mouse heart. To investigate ssTnI downregulation further, cTnI knockout mice were used to examine a possible role for thyroid hormone. Northern blot analysis of euthyroid animals showed a time-dependent loss of ssTnI mRNA that was similar for wild-type, heterozygous and homozygous cTnI mutant mice. In cTnI null mice made hyperthyroid by l -thyroxine, the duration of ssTnI expression assessed by both mRNA and protein content was abbreviated compared with the euthyroid group. Hyperthyroid cTnI null mice also died significantly earlier than euthyroids (postnatal day 14 v day 18). In cTnI null mice made hypothyroid by addition of phenylthiouracil to the drinking water, ssTnI expression was prolonged and mice survived until day 20 or 21. Overall, the results indicate that inactivation of the ssTnI gene occurs even in the absence of cTnI mRNA and protein indicating that these are not critical signals for ssTnI down regulation in the heart. In contrast, thyroid hormone influences the time course of ssTnI expression and the life span of cTnI null mice probably through a transcriptional regulation of ssTnI in the heart.

Vigabatrin-associated visual field defects in children.

PURPOSE: Vigabatrin (Sabril), a drug that blocks GABA transaminase, has been used in the treatment of epilepsy since 1989. There have been reports of irreversible constriction of the visual field in adult patients related to vigabatrin (VGB) therapy, resulting in reduced VGB usage in adults. Although used as a second or third line agent in adults, in children it is often considered as a first line treatment for several subgroups of seizures in spite of there being no way, in the majority of cases, to monitor visual fields. Some of these children have a pre-existing visual field defect as part of their primary disorder. We aimed to identify whether visual field loss due to VGB was occurring in our hospital. METHODS: We have studied the results of ophthalmic examination in 14 children on VGB at Great Ormond Street Hospital who were able to perform Goldmann visual fields. RESULTS: Ten of the 14 patients had constriction of their visual fields attributed to VGB. In addition there were 2 patients with suspicious visual field defects thought to be due to VGB. There was pre-existing visual pathway damage in 4 cases and in 2 of these optic disc pallor increased in association with constricted visual fields. However, the optic discs were normal in 7 patients in spite of visual field constriction. Visual acuity was generally normal in spite of gross visual field constriction. CONCLUSIONS: We believe that VGB should be used with great caution where there is pre-existing visual pathway damage. In other cases the benefits should be considered in relation to the risks, which include irreversible visual field damage. At present visual fields can only be monitored by perimetry, which is often not possible in children with epilepsy.

Diacylglycerol and fatty acids synergistically increase cardiomyocyte contraction via activation of PKC.

Lipid signaling pathways are thought to play a prominent role in transducing extracellular signals into contractile responses in cardiac muscle. Two putative lipid messengers, diacyglycerol and arachidonic acid, can be generated via distinct phospholipases in separate signaling pathways, but certain stimuli cause them to be elevated in parallel. We tested the hypothesis that these lipids function as comessengers in ventricular myocytes by activating protein kinase C (PKC). In previous work, we demonstrated that the diacylglycerol analog dioctanoylglycerol (diC(8)) can be stimulatory or inhibitory toward myocyte twitches depending on how it is applied. Here we report that arachidonic acid and other cis-unsaturated fatty acids (UFA), at concentrations too low for direct effects, synergistically enhance the stimulatory effects of diC(8) and convert inhibitory effects of diC(8) into stimulation of myocyte twitches. Intracellular Ca(2+) transients changed in parallel with twitch amplitude, suggesting regulation of Ca(2+) homeostasis by these lipids. cis-UFA also interacted synergistically with the PKC activator phorbol 12-myristate 13-acetate to promote positive inotropic responses. Responses were blocked by the PKC antagonists chelerythrine chloride, bisindolylmaleimide, and Gö-6976. DiC(8) and arachidonic acid also synergistically translocated PKC-epsilon and PKC-alpha in intact myocytes. We propose that PKC integrates diacylglycerol and cis-UFA signals in the heart, resulting in preferential activation of positive inotropic mechanisms.

Endothelin-1 and photoreleased diacylglycerol increase L-type Ca2+ current by activation of protein kinase C in rat ventricular myocytes.

The amphotericin B-perforated whole-cell patch clamp technique was used to determine the modulation of L-type Ca2+ channels by protein kinase C (PKC)-mediated pathways in adult rat ventricular myocytes. Application of 10 nM endothelin-1 (ET-1) increased peak Ca2+ current (ICa) by 28.2 +/- 2.5 % (n = 13) and slowed current decay. These effects were prevented by the endothelin receptor antagonist PD145065 (10 microM) and by the PKC inhibitor chelerythrine (8 microM). To establish if direct activation of PKC mimicked the ET-1 effect, the active and inactive phorbol esters (phorbol-12-myristate-13-acetate and 4alpha-phorbol-12, 13-didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (approximately 10%) increase in ICa, suggesting PKC-independent effects. Bath application of dioctanoylglycerol (diC8), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak ICa (50.4 +/- 6.2 %, n = 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 microM). Intracellular photorelease of caged diC8 with 3 or 10 s exposure to UV light produced a concentration-dependent increase in peak ICa (20. 7 +/- 8.5 % (n = 8) for 3 s UV and 60.8 +/- 11.4 % (n = 13) for 10 s UV), which could be inhibited by chelerythrine. Our results demonstrate that both ET-1 and intracellularly photoreleased diC8 increase ICa by a PKC-mediated pathway, which is in direct contrast to the PKC-independent inhibition of ICa produced by bath-applied diC8. We conclude that specific cellular pools of DAG are crucially important in the regulation of ICa by PKC.

Impaired calcium release in cerebellar Purkinje neurons maintained in culture.

Cerebellar Purkinje neurons demonstrate a form of synaptic plasticity that, in acutely prepared brain slices, has been shown to require calcium release from the intracellular calcium stores through inositol trisphosphate (InsP(3)) receptors. Similar studies performed in cultured Purkinje cells, however, find little evidence for the involvement of InsP(3) receptors. To address this discrepancy, the properties of InsP(3)- and caffeine-evoked calcium release in cultured Purkinje cells were directly examined. Photorelease of InsP(3) (up to 100 microM) from its photolabile caged analogue produced no change in calcium levels in 70% of cultured Purkinje cells. In the few cells where a calcium increase was detected, the response was very small and slow to peak. In contrast, the same concentration of InsP(3) resulted in large and rapidly rising calcium responses in all acutely dissociated Purkinje cells tested. Similar to InsP(3), caffeine also had little effect on calcium levels in cultured Purkinje cells, yet evoked large calcium transients in all acutely dissociated Purkinje cells tested. The results demonstrate that calcium release from intracellular calcium stores is severely impaired in Purkinje cells when they are maintained in culture. Our findings suggest that cultured Purkinje cells are an unfaithful experimental model for the study of the role of calcium release in the induction of cerebellar long term depression.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors.

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor.

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.