Australian surveys on incidence and control of blowfly strike in sheep between 2003 and 2019 reveal increased use of breeding for resistance, treatment with preventative chemicals and pain relief around mulesing.

Blowfly strike or cutaneous myiasis, caused principally by the Australian sheep blowfly Lucilia cuprina is endemic to Australian sheep producing areas and is a major cause of suffering and economic loss. This article reports incidence and control practices as determined by three cross-sectional surveys of Australian sheep farmers covering the years 2003 (n = 1365), 2011 (n = 575) and 2018 (n = 354) with more questions relating to blowfly strike in the latter two surveys. Breech strike was reported at a higher rate than body strike in all survey years. Reported annual incidence of breech strike ranged from 2.2 to 4.7% of sheep nationally with minor differences between ewes and lambs. The incidence of body strike ranged from 1.0 to 7.1% with higher incidence in younger sheep in all years. The use of fixed time routine preventative chemical treatments increased during the survey period (43%, 46% and 66%, in 2003, 2011 and 2018 respectively) with dicyclanil (54%-58%) and cyromazine (23-36%) the main insecticides used for control since 2011. The use of the Mules operation (mulesing) to remove skin wrinkles from the breech area as a preventative modification did not change between 2011 and 2018 (47% in both), however, the use of pain relief at mulesing of replacement ewe lambs increased substantially between 2011 (59%) and 2018 (87%). The most commonly used methods to assist with flystrike control were timing of crutching (clipping of wool from susceptible areas) and shearing, with few respondents destroying maggots from treated sheep (21-26%) and very low use of fly traps (5-8%). Phenotypic selection of ewes for reduced susceptibility to flystrike based on visual traits was practiced by 61% of respondents in 2011 and 56% in 2018. Selection of rams for reduced susceptibility using estimated breeding values increased from 10% in 2011 to 17% in 2018. Breeding for resistance was the most commonly nominated (21%) most important change made to flystrike management in 2018. The results indicate that concern for sheep welfare has increased with almost universal use of pain relief at mulesing. Increased use of selection for blowfly resistance indicates farmer commitment to planning for a future without mulesing, although the practice remains prevalent. An increase in the reliance on preventative chemical treatments increases the risk of insecticide resistance.

Prevalence of sheep lice and trends in control practices across Australia - Australian sheep parasite control surveys from 2003 to 2019.

The sheep body louse (Bovicola ovis) is a parasite of major economic concern in Australia. This article reports lice incidence and control practices on Australian sheep farms as determined by three cross-sectional surveys of the years 2003, 2011 and 2018. The incidence of live lice detected within a flock was similar across the survey years with a slight decline in 2018 (2003: 20%, 2011: 18.6% and 2018: 13.9%). In an average year, most farmers did not see any direct or indirect evidence of lice infestation (2011: 59.3% and 2018: 69.5%), however, over a five-year period 38.9% (2003), 66.3% (in 2011) and 55.8% (in 2018) reported seeing evidence of lice infestation at least once with significant variation between sheep producing regions in 2011. In an average year, nearly three quarters of respondents treated for lice (2011:73.5% and 2018: 73.3%) with the majority treating immediately following shearing and very few treating sheep with long wool. Even higher percentages treated for lice at least once in the preceding five-year period (2011: 86.2% and 2018: 87.5%). Backliner was the most popular method of chemical application for lice control in sheep off-shears or with short wool (2003: 77%, 2011: 73% and 2018: 74.3%). For long wool treatments, hand jetting declined in popularity from 2003 (64%) to 2011 (54%) and 2018 (8.6%) as backliners became more popular (2003: 36%, 2011: 51% and 2018: 60%). The use of benzoylphenyl urea insect growth regulators (IGR) for off-shears/short wool treatment declined from 2003 (92.8%) to 2011 (51%) and 2018 (2.9%) and were largely replaced by neonicotinoids and spinosad for backliner/spray-on products. The use of organophosphates declined for plunge dipping (2003: 83.8%, 2011: 83% and 2018: 7.7%). Spinosad use for plunge dipping off-shears/short wool increased over the survey years (2003, 0%, 2011: 9% and 2018:46.2%). The use of IGRs declined for backliner application on long wool and were mainly replaced by spinosad in 2011 and 2018. Fewer respondents reported suspected resistance to lice control products in 2018 (8%) compared with 2003 (26%) and 2011 (13%) with most reporting suspected resistance to IGR and synthetic pyrethroids and emerging suspicions of resistance to neonicotinoids in 2018. Resistance to lice control products also reduced in importance as a reason for recurring lice infestations between 2011 (ranked 2nd) and 2018 (ranked 6th). Biosecurity was important to sheep producers with the highest ranked reason for recurring lice infestations being from stray or purchased sheep.

Benchmarking Australian sheep parasite control: Changes in gastrointestinal nematode control practices reported from surveys between 2003 and 2019.

Cross-sectional surveys of sheep parasite control practices in Australia were conducted in 2004, 2012 and 2019 to document parasite problems, control practices and measure change over time. This article reports the results pertaining to gastrointestinal nematode infection; comparisons between years are mostly descriptive and not based on statistical inference. There was a general increase in the use of grazing management to prepare clean pastures for sheep to control gastrointestinal nematode infections with the largest increases in the use of: cropping, long acting anthelmintics to prepare clean pastures, feeding strategies, selecting rams for resistance to nematodes, and leaving some sheep untreated. The proportion of respondents using faecal worm egg count monitoring (WEC) and the number of WEC monitors per year were similar in 2003 (weaners: 3.0 WEC/year, adult ewes: 2.6 WEC/year) and 2018 (lambs and weaners and adult ewes both 3.1 WEC/year) but lower in 2011(lambs and weaners: 2.0 WEC/year, adult ewes: 2.9 WEC/year), whilst there was a higher number of anthelmintic treatments given in 2011 than 2003 and 2018 which were similar. There was an increase in the proportion of respondents carrying out WEC monitors themselves from 2011 (21%) to 2018 (30%) and a corresponding reduction in the use of private laboratories, government laboratories and veterinarians with regional differences in who carried out WECs. The top three anthelmintic groups used did not differ between the three survey years with macrocyclic lactones the most used followed by benzimidazoles (BZ) and levamisole (LEV) although the percentage of MLs used in 2011 and 2018 was lower than in 2003 with higher proportions of respondents using BZ and LEV groups in the latter two survey years. Moxidectin was in the most commonly used active in all survey years. There was an increase in the use of combination of anthelmintics from different groups, especially for a combination of three anthelmintics (2003: 4.4%, 2011: 19.1%, 2018: 21.5%), with lower use of single anthelmintics in 2011 (57.0%) and 2018 (55.4%) compared with 2003 (74.5%). The use of testing for anthelmintic resistance was generally low across the survey years with a lower proportion of respondents using tests in 2011 and 2018 than in 2003 (2003:48%, 2011: 29%, 2018: 37%). Time of year, results from WEC and seasonal weather condition were the most important factors when deciding when to apply anthelmintic treatment.

Development of a postoperative care pathway for children with renal tumors.

PURPOSE: To identify the factors associated with a shorter postoperative stay, as an initial step to develop a care pathway for children undergoing extirpative kidney surgery. STUDY DESIGN: This study retrospectively reviewed patients managed with upfront open radical nephrectomy for renal tumors between 2005 and 2016 at a pediatric tertiary care facility. Univariate and multivariate logistic regression were performed to identify factors associated with early discharge (by postoperative day 4). RESULTS: A total of 84 patients met inclusion criteria. Median age was 28.1 months (range 1.8-193.1). Thirty-four (40.5%) patients had a nasogastric tube postoperatively. The patients were advanced to a clear liquid diet on a median postoperative day 2 (range 0-7) and regular diet on a median postoperative day 3 (range 1-8). Median time from surgery to discharge was 5 days (range 2-12), with 38 (45.2%) discharged early. Univariate and multivariate logistic regression analyses showed that earlier resumption of regular diet (OR 0.523, P = 0.028) was positively associated with early discharge. Other analyzed factors were not significant (see Table). DISCUSSION: Timely initiation of adjuvant therapy is a specific requirement of Children's Oncology Group (COG) protocols. Chemotherapy and radiation therapy are ideally initiated simultaneously, as early as possible, within 2 weeks of surgery. Thus, factors that can facilitate early discharge from the hospital can maximize protocol adherence with respect to timing of adjuvant therapy initiation and optimize patient outcome. This study shed light on several postoperative factors and how these relate to postoperative stay and recovery. Specifically, tumor size, pre-operative bowel preparation, extent of lymph node sampling, stage, operative time, estimated blood loss, surgical service, postoperative nasogastric tube use, transfusion, and chemotherapy prior to discharge were not associated with discharge timing. Early re-feeding was associated with early discharge. Thus, it seems reasonable that, when developing a postoperative care pathway for these patients, these factors be considered and specifically encourage early re-feeding. In pediatrics, data on early recovery after surgery protocols are limited, and high-quality studies are unavailable. Within pediatric urology, early recovery after surgery protocols in children undergoing major urologic reconstruction have been shown to reduce hospital stay and can decrease complication rates. It seems reasonable that a similar pathway can be applied to children undergoing radical nephrectomy for suspected malignancy. CONCLUSIONS: For children with renal tumors who underwent radical nephrectomy, early re-feeding was associated with a shorter time to discharge. Use of bowel preparation and nasogastric tube did not appear to shorten time to discharge. These data are important for developing postoperative care pathways for these patients.

Role of host and environment in mediating reduced gastrointestinal nematode infections in sheep due to intensive rotational grazing.

We have previously reported marked reductions in faecal worm egg counts (WECs) and drenching frequency in sheep on an intensive rotational grazing system (IRG) in a cool temperate environment with summer-dominant rainfall. These experiments were designed to determine the role of the host and environmental factors in mediating this. The role of host factors was investigated by administering a fixed larval challenge in each of the 4 seasons of the year to groups of 20 young sheep on three different management systems, including IRG. This comprised a mixed larval challenge containing infective larvae of Haemonchus contortus and Trichostrongylus colubriformis 7 days after short-acting anthelmintic treatment. A range of measurements was then made up to day 35 post-challenge. The role of environmental factors was determined by assessing pasture infectivity in four seasons using faecal worm egg counts (WECs) and pooled faecal culture of worm-free tracer sheep. The management systems were high input (HI) with high fertiliser inputs high stocking rate and relatively long grazing periods; typical New England management system (TYP) with moderate fertiliser inputs and stocking rate and relatively long grazing periods and; Intensive rotational grazing (IRG) with moderate fertiliser inputs and stocking rate but very short (mean 5 days) grazing periods and long (mean 103 days) rest periods. IRG sheep had higher mean WEC at 28 and 35 days after fixed larval challenge than HI and TYP sheep in spring (IRG: 9500 ± 1000; HI: 4000 ± 1000; TYP: 7200 ± 1000 eggs/g, P<0.01) and summer (IRG: 8400 ± 750; HI: 5300 ± 800; TYP: 4400 ± 700 eggs/g; P<0.001) and also had lower live weights during these seasons. There was no difference in WEC after the autumn challenge (IRG: 5100 ± 450 HI: 4500 ± 450; TYP: 4200 ± 450 eggs/g; P ≈ 0.36) but IRG had lower WEC than TYP following the winter challenge (IRG: 2900 ± 400; HI: 2300 ± 400; TYP: 4300 ± 400 eggs/g, P<0.01). The tracer sheep (used to determine pasture infectivity) on IRG had significantly lower WECs during winter, spring and summer than those under the other management systems. Faecal culture and larval differentiation revealed that faeces from tracers on IRG contained significantly lower proportions of H. contortus and significantly higher proportions of Trichostrongylus spp. and Teladorsagia circumcincta than faeces from tracers on the HI and TYP treatments. Thus, when IRG was most efficacious for worm control, during spring and summer when short graze and long rest periods were maintained, sheep on this system exhibited greater susceptibility to larval challenge while tracer sheep indicated lower pasture infectivity. This demonstrates that the effects of IRG on WEC are mediated by reduced larval challenge rather than increased host resistance to infection.

Intensive rotational grazing assists control of gastrointestinal nematodosis of sheep in a cool temperate environment with summer-dominant rainfall.

While rotational grazing methods have an accepted role in the management of gastrointestinal nematodosis (GIN) of small ruminants in humid tropical regions, their efficacy and application in cool temperate regions is more controversial. This study evaluated GIN over 2 years in three classes of fine wool Merino sheep (lambs, hoggets and ewes) under three different sheep management systems in a cool tableland environment near Armidale NSW Australia (950m altitude, 30 degrees 31'S, 151 degrees 39'E). The management systems were High input (HI) with high fertiliser inputs, a target of 100% sown pasture, high stocking rate (13.4 dry sheep equivalents/ha) and relatively long grazing periods; Typical (TYP) New England management system with moderate fertiliser inputs and stocking rate (9.3DSE/ha) and relatively long grazing periods; and Intensive rotational grazing (IRG) with moderate fertiliser inputs and stocking rate (8.8DSE/ha) but very short (mean 5 days) grazing periods and long (mean 103 days) rest periods. Twenty sheep of each class in each management treatment were sampled monthly for faecal worm egg counts (WEC, followed by larval differentiation), and body weights with a blood sample taken for haematology every second month. The proportion of sheep with WEC above zero did not differ between management systems but the magnitude of WEC did, with sheep under IRG displaying lower mean WEC than those on the other treatments (IRG: 326, HI: 594, TYP: 536, eggs/g P<0.0001). This was despite a significantly longer mean interval between anthelmintic treatments (IRG: 144 days, HI: 77 days, TYP: 78 days, P<0.0001). The IRG management system also influenced the composition of the infections with sheep on this treatment having a significantly lower proportion of Haemonchus contortus in their faecal cultures (IRG: 59.7%, HI: 79.4%, TYP: 80.9%, P<0.05) and a significantly higher proportion of Trichostrongylus spp. Sheep on the IRG treatment also had a significantly higher haematocrit (HCT) than those on the other management systems. Despite the lower WEC and higher HCT, sheep under IRG also had significantly lower bodyweights and fleece weights overall, although this was only evident in sheep raised to adulthood prior to the experiment, not those raised during the 2-year experimental period. The results demonstrate that IRG systems with short grazing periods and long rest periods between grazing events can assist with control of GIN in cool temperate climates where H. contortus is the dominant parasite.

A normative study of levels of uranium in the urine of British Forces personnel.

OBJECTIVES: The UK Ministry of Defence (MoD) controls a biological monitoring programme that includes testing for uranium in personnel who served in the conflict in Iraq in 2003. To help interpret the results, the MoD commissioned this study to quantify a normative reference distribution of urinary uranium concentrations in military personnel who had not served in that conflict. METHODS: The study selected and visited various military establishments to recruit a representative mix of ranks, genders and occupational groups (combat, support and auxiliary). A standardised protocol and recruitment questionnaire were used. The 125 ml spot urine samples collected were analysed for uranium and creatinine concentrations and (where possible) for uranium isotope (238)U/(235)U ratio. RESULTS: Samples from 732 eligible subjects were analysed. Adjusted uranium concentrations ranged up to 556 ng.g(-1) creatinine, somewhat higher than reference values quoted for the USA but much lower than recorded in granite areas such as Finland. Isotope ratio measurements were available for 125 samples (17%) with the highest concentrations; these all had a natural isotope signature and no evidence of depleted uranium (DU). On average, urinary uranium concentrations were somewhat lower in officers than in other ranks; they differed also across the services, the Navy being lowest and the Army highest. The levels give no concern regarding health risks in the personnel studied. CONCLUSION: Since even the highest values were from natural sources, we assume the differences represent differences in ingestion of natural uranium. Definition of a reference distribution or normal values will depend on the subpopulation of interest.

A normative value pilot study: levels of uranium in urine samples from UK civilians.

A normative study of the levels of urinary uranium in the general UK population is needed for comparison with levels in UK military and ex-military personnel who served where munitions containing depleted uranium (DU) were used. As preparation, this pilot study trialled the process of collecting 24-h samples from adult male civilians, and compared the measurements from 24-h samples with those from spot samples taken over the subsequent 24h. The purpose was to assess the relative utility of the two types of samples. Twenty-five convalescent hospital in-patients were recruited as participants. Uranium concentrations in the 24-h samples ranged from 1 to 10.6 ng l(-1); in the spots, from not detectable to 38.1 ng l(-1). Normalised to creatinine, concentrations in the 24h samples ranged from approximately 100 to 800 ng mol(-1) creatinine; in the spot samples, from not detectable to approximately 4000 ng mol(-1) creatinine. The ranges appear similar to those reported for residents of the US. The distribution of spot sample results indicated that 95% of a participant's creatinine-adjusted concentrations from spot samples would be within the range 40-250% of his mean. Adjusting for creatinine almost entirely eliminated a slight indication of diurnal variation in urinary uranium concentration in spot samples. All the 24-h samples and 131 out of the 133 spot samples showed ratios of isotopes (238)U to (235)U consistent with natural uranium (i.e. neither enriched nor depleted). Slightly elevated ratios in two spot samples were not supported by other samples from the same participants, indicating that slightly elevated ratios may be recorded on very low concentration (<1 ng l(-1)) samples. In the main, quantification of this isotope ratio from spot samples was only slightly more variable than from 24-h samples. Complete 24-h urine samples gave better precision than spot samples in estimating uranium concentrations at these low levels, but presented more logistic difficulties in the collection of the samples. Clarification of the relative merits of alternative sampling strategies enables the design of a wider study to be optimised.

Laparoscopic anterior esophageal myotomy and toupet fundoplication for achalasia.

Achalasia is an esophageal motility disorder characterized by the failure of lower esophageal sphincter relaxation and the absence of esophageal peristalsis. The purpose of this study was to evaluate the clinical outcomes of patients undergoing laparoscopic esophageal myotomy and Toupet fundoplication for achalasia. A 9-cm myotomy was performed in most cases extending 7 cm above and 2 cm below the gastroesophageal junction. Severity of dysphagia, heartburn, chest pain, and regurgitation was graded preoperatively and postoperatively using a five-point symptomatic scale (0-4). Patients also graded their outcomes as excellent, good, fair, or poor. Between December 1995 and November 2000 a total of 49 patients (23 male, 26 female) with a mean age of 44.3 years (range 23-71 years) were diagnosed with achalasia. Mean duration of symptoms was 40.2 months (range 4-240 months). Thirty-seven patients (76%) had had a previous nonsurgical intervention or combinations of nonsurgical interventions [pneumatic dilation (23), bougie dilation (five), and botulinum toxin (19)], and two patients had failed esophageal myotomies. Forty-five patients underwent laparoscopic esophageal myotomy and Toupet fundoplication. Two patients received laparoscopic esophageal myotomies without an antireflux procedure, and two were converted to open surgery. One patient presented 10 hours after a pneumatically induced perforation and underwent a successful laparoscopic esophageal myotomy and partial fundoplication. Mean operative time was 180.5 minutes (range 145-264 minutes). Mean length of stay was 1.98 days (range 1-18 days). There were five (10%) perioperative complications but no esophageal leaks. There was a significant difference (P < 0.05) between the preoperative and postoperative dysphagia, chest pain, and regurgitation symptom scores. All patients stated that they were improved postoperatively. Eighty-six per cent rated their outcome as excellent, 10 per cent as good, and 4 per cent as fair. Laparoscopic anterior esophageal myotomy and Toupet fundoplication effectively alleviates dysphagia, regurgitation, and chest pain accompanying achalasia and is associated with high patient satisfaction, a rapid hospital discharge, and few complications.

Motoric behavior in aged rats treated with GM1.

Aging is associated with impaired motor function. Nigrostriatal dopaminergic neurons, in part, regulate motoric behavior, and undergo degenerative changes during aging. GM1 ganglioside partially restores pre-synaptic dopaminergic markers and the number and morphology of dopaminergic neurons in the midbrain and striatum of Sprague--Dawley aged rats. These studies investigated whether GM1 treatment, 30 mg/kg, i.p. daily for 36 days, affects locomotor and stereotypic activity, as well as coordination, balance, and strength in aged rats. Under the treatment conditions used, GM1 did not improve the reduced locomotor and stereotypic behavior of the aged rats. While it partially improved performance on a square bridge test, GM1 had no effect on inclined screen and rod suspension tests. Although GM1 restored the decreased content of dopamine and homovanillic acid in the nigrostriatal neurons of the aged rats, it had no effect on the reduced D1 and D2 dopamine receptor binding and mRNA in the striatum. It appears, that despite the morphological and metabolic restoration of aged nigrostriatal neurons, GM1 has limited ability in improving age-associated motor deficits.

[Comparison of Trypanosoma cruzi infection in less than 10 years old children from the V Región, Chile, 1982-1995]. / Comparación de la infección por Trypanosoma cruzi en niños menores de 10 años de la V región, Chile. 1982-1995.

Evaluation of desinsectation programs carried out in the two Health Services from the V Region, was undertaken by comparing serologic prevalences of T. cruzi infection age groups exposed to the risk of infection in rural areas during the antivectorial campaign activities (1982 vs 1995). Thus, were studied 2,193 blood samples from children under 10 years of age, proceeding from six chagasic endemic provinces in which antitriatomic domiciliary insecticide sprayings had been performed. Indirect hemagglutination and ELISA tests were carried out to each of the blood samples. A total de 42 (1.9%) children resulted positive. As in five counties no positive cases were detected in the last five years it is possible to assume that vector transmission of T. cruzi should have been interrupted in them. When comparing prevalences existing in 1982 with the present ones, it is possible to observe a 63.5% of reduction of transmission in the V Region, been this reduction higher in the Aconcagua Health Service with 79.6% and lower in the Viña del Mar--Quillota Health Service with 55.6%. According to these results, comparison of prevalence of T. cruzi infection in children less than 10-year-old in diverse periods, allows evaluate the vectorial control of Triatoma infestans programs.

Block of brain sodium channels by peptide mimetics of the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate.

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH2, measured on sodium channels whose inactivation was slowed by the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH2 did not cause appreciable fast block, but the divalent cation IFM-NH(CH2)2NH2 was as effective as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2 showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.

cAMP-dependent phosphorylation of two sites in the alpha subunit of the cardiac sodium channel.

The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.

Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.

Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.

Differential proteolysis of the full-length form of the L-type calcium channel alpha 1 subunit by calpain.

This study examines the proteolysis of the carboxy terminal domain of the full-length (alpha 1(212)) and truncated (alpha 1(190)) forms of the rabbit skeletal muscle L-type calcium channel alpha 1 subunit by calpain I and calpain II. Although both forms of the alpha 1 subunit show little sensitivity to proteolysis by calpain II, alpha 1(212) is relatively more sensitive than alpha 1(190) to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of alpha 1(212) in a time- and concentration-dependent manner and proteolysis is independent of the alpha 1(212) phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from alpha 1(212) and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.

Individual differences in basal and cocaine-stimulated extracellular dopamine in the nucleus accumbens using quantitative microdialysis.

The current experiment examined the role of nucleus accumbens (NACC) dopamine in individual differences. Subjects were divided into high responders (HR) and low responders (LR) based on their locomotor response to a novel environment. HR rats were subjects which had a locomotor response to novelty in the upper third of the population screened and LR rats in the bottom third of the population. A new method of microdialysis was then used that allowed determination of the extracellular dopamine concentration. This was accomplished by adding various dopamine concentrations (0.0, 5.0 and 20.0 nM) to the perfusate. The concentration of dopamine in the dialysate was subsequently determined. The difference in the dialysate and perfusate dopamine was regressed on the perfusate dopamine. The regression yielded the in vivo recovery and the extracellular concentration. HR rats exhibit a 250% higher basal dopamine concentration (6.45 +/- 1.01 nM, n = 6) than LR rats (2.58 +/- 0.16 nM, n = 7). The in vivo microdialysis recovery was used to estimate the extracellular dopamine following cocaine challenge (15 mg/kg) in the two groups. Following i.p. cocaine administration, HR rats had both a greater locomotor response and increase in absolute dopamine concentration compared to LR rats. The maximum dopamine concentration in the HR group was 23 +/- 2.9 nM, while that in the LR group was only 8.6 +/- 1.1 nM. The maximum in the LR group is comparable to the basal level in the HR group. However, there were no difference in percent change in dopamine following cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of cabbage proteinase inhibitorsin situ on the growth of larvalTrichoplusia ni andPieris rapae.

Trypsin and chymotrypsin inhibitors are proteins that are developmentally regulated in foliage of cabbage plants, appearing at high concentrations in young foliage on mature plants. This temporal and spacial regulation of foliar proteinase inhibitors is synchronized with the appearance and distribution of foliar feeding Lepidoptera. When insects were allowed to select their feeding sites, larvalPieris rapae fed on the young foliage of cabbage plants, while larvalTrichoplusia ni fed on the mature foliage on cabbage plants. LarvalP. rapae that fed on mature plants were significantly smaller than larvae feeding on young plants, while there was no significant difference between larvalT. ni feeding on mature plants and those feeding on young plants. Thus, there was a significant inverse correlation between the level of proteinase inhibitory activity in cabbage foliage and larval growth. WhenP. rapae andT. ni were provided with an artificial diet containing total protein (including significant levels of proteinase inhibitors) that was extracted from cabbage foliage, there was a significant reduction in growth and development of both species of Lepidoptera.