Behavioral and Physiological Reactions to a Sudden Novel Object in the Weanling Horse: Quantitative Phenotypes for Future GWAS.

The startle response can be defined as a reflexive reaction to the sudden appearance of a novel stimulus that influences the survival and resilience of animals. In domesticated species, the behavioral component of the startle response can, in some cases, cause serious injury to the animal or human handlers if inappropriately expressed. Here, we describe a longitudinal study in a population of stock-type horses that quantified behavioral startle responses elicited by the presentation of a sudden novel object (rapidly opening umbrella). The study was performed in weanling foals across four consecutive years (n = 74, mean age = 256 days). Behavioral assays for the startle response phenotype focused on six behavioral variables: latency to return to the feed pan (seconds), maximum distance fled (meters), proportion of time spent walking or trotting (seconds), and how long a horse spent standing facing away from or toward the novel object. We observed behavioral startle response variables in relation to cardiac response, age, and sex for each individual. Each horse's cardiac startle response pattern was determined and categorized into heart rate response cluster groups identified as accelerators and decelerators. Using principal component analysis (PCA) with a factor rotation, we identified "startle response" phenotypes that summarize the behavioral and physiological variables. The largest component of variation, Factor 1, comprised 32.5% of the behavioral variable with a positive correlation with latency and distance, and was not influenced by sex or age. Factor 2 comprised 23.2% of the variation, and was positively correlated with activity level performed such as proportion of time spent walking and/or trotting. Horses with the accelerator type cardiac response had significantly higher Factor 1 scores than decelerators but did not differ in Factor 2. Future work includes expanding our sample size to conduct a genome-wide association study (GWAS) to identify novel genetic loci influencing behavioral startle reactions using recorded behavioral and physiological phenotypes.

Culinary Nutrition Education Programs in Community-Dwelling Older Adults: A Scoping Review.

BACKGROUND: Culinary nutrition education programs are increasingly used as a public health intervention for older adults. These programs often integrate nutrition education in addition to interactive cooking workshops or displays to create programs suitable for older adults' needs, ability and behaviour change. Synthesising the existing literature on nutrition education and interactive cooking programs for older adults is important to guide future program development to support healthy ageing. OBJECTIVES: To determine the extent of published literature and report the characteristics and outcomes of interactive culinary nutrition education programs for older adults (> 51 years). DESIGN: This scoping review followed the PRISMA-ScR guidelines recommended for reporting and conducting a scoping review. METHODS: Five databases were searched of relevant papers published to May 2022 using a structured search strategy. Inclusion criteria included: older adults (≥ 51 years), intervention had both an interactive culinary element and nutrition education and reported dietary outcome. Titles and abstracts were screened by two reviewers, followed by full-text retrieval. Data were charted regarding the characteristics of the program and outcomes assessed. RESULTS: A total of 39 articles met the full inclusion criteria. The majority of these studies (n= 23) were inclusive of a range of age groups where older adults were the majority but did not target older adults exclusively. There were large variations in the design of the programs such as the number of classes (1 to 20), duration of programs (2 weeks to 2 years), session topics, and whether a theoretical model was used or not and which model. All programs were face-to-face (n= 39) with only two programs including alternatives or additional delivery approaches beside face-to-face settings. The most common outcomes assessed were dietary behaviour, dietary intake and anthropometrics. CONCLUSION: Culinary nutrition education programs provide an environment to improve dietary habits and health literacy of older adults. However, our review found that only a small number of programs were intentionally designed for older adults. This review provides a summary to inform researchers and policy makers on current culinary nutrition education programs for older adults. It also recommends providing face-to-face alternatives that will be accessible to a wider group of older adults with fewer restrictions.

A rapid assay for mitochondrial DNA damage and respiratory chain inhibition in the yeast Saccharomyces cerevisiae.

There is a need for a rapid assay to identify agents that damage mitochondria because the mitochondrion may be an important target for numerous environmental mitotoxins. Certainly at least one chemotherapeutic regimen (CHOP therapy) that includes doxorubicin can induce cardiomyopathy through mitochondrial genotoxicity in cardiac muscle cells. Yeast cells (1.5 x 10(6)-10(7)) in water are spread on a YEPD plate, and, when the suspension of cells has dried, a small well (12 mm diameter) is cut into the agar; 200-400 microl of a solution of the presumptive mitochondrial genotoxin is placed in the well, and the plates are incubated for 2 days. The genotoxin forms a concentration gradient through the agar and affects the growing cells. An overlay containing tetrazolium chloride is added, and the plates are incubated for 6-24 hr. Respiring cells turn red, and nonrespiring cells, with damaged DNA or inhibited respiratory chains, that are adjacent to the well, are white. A white ring, or a more lightly colored red ring, around the well indicates the presence of cells with lowered respiratory activity which may be fully reversible when the mitochondrial genotoxin is removed. In preliminary experiments, doxorubicin (= adriamycin) shows strong activity with this assay; cyclophosphamide is negative, and 4-hydroxycyclophosphamide, a metabolite of cyclophosphamide, is weakly positive. Ethidium bromide, methotrexate, 5-fluorouracil, and 5-fluorocytosine also are mitochondrial genotoxins. Antifungal agents similar to 5-fluorocytosine and anthelmintic compounds such as pyrvinium iodide can be powerful mitochondrial genotoxins.

Protective effects of valsartan and benazeprilat in salt-loaded stroke-prone spontaneously hypertensive rats.

These experiments examined the effectiveness of chronic blockade of the renin angiotensin system with either valsartan or benazeprilat on survival, blood pressure and end-organ damage in salt-loaded stroke-prone SHR. Valsartan or benazeprilat given continuously by subcutaneous osmotic minipump beginning at 10.5 weeks of age lowered blood pressure, as determined by radiotelemetry, prevented proteinuria, prolonged survival and decreased the severity of histopathological changes in the heart and kidney. These results indicate that angiotensin receptor blockade affords a similar degree of protection as inhibition of angiotensin converting enzyme in salt-loaded stroke-prone SHR. Furthermore, our results are consistent with a primary contribution of angiotensin II to the maintenance of blood pressure and support a principal role for angiotensin II-dependent mechanisms in the development of end-organ damage in the salt-loaded stroke-prone SHR.

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

Cardiovascular effects of adenosine A2 agonists in the conscious spontaneously hypertensive rat: a comparative study of three structurally distinct ligands.

The hemodynamic responses to 5'-N-ethylcarboxamide adenosine (NECA), a nonselective adenosine agonist, were compared to those elicited by the sodium salt of 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine (CGS 21680C) and N6-2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl adenosine (CGS 24012), two structurally dissimilar selective A2 agonists in conscious spontaneously hypertensive rats (SHR). Dose-related reductions in mean arterial pressure occurred after bolus administration of NECA, CGS 21680C and CGS 24012. Dose-dependent tachycardia was seen with both CGS 21680C and CGS 24012, whereas NECA produced a biphasic response on heart rate. At high doses (3 and 10 micrograms/kg), NECA evoked an immediate and dramatic fall in heart rate, followed by a more gradual and long-lasting tachycardia. At equihypotensive doses, both CGS 21680C and CGS 24012 produced significant increases in cardiac output, but NECA had no effect. Although each of the adenosine agonists reduced total peripheral resistance, the greatest change was produced by CGS 21680C. Hindquarter, renal and mesenteric vascular resistances were significantly reduced by both CGS 21680C and CGS 24012, whereas only mesenteric vascular resistance was reduced with NECA. CGS 24012 reduced renal vascular resistance to the greatest extent and produced a concomitant significant increase in renal blood flow. Marked elevation in plasma renin activity occurred with CGS 24012 and CGS 21680C, whereas no change was seen after NECA. The hemodynamic responses to NECA, CGS 21680C and CGS 24012 were significantly reduced by the adenosine antagonist, 8-(p-sulfophenyl) theophylline, suggesting that these agents act through stimulation of adenosine receptors in the conscious SHR. Furthermore, blockade of the beta adrenergic receptor with metoprolol (1 mg/kg, i.v.) significantly attenuated the increase in heart rate produced by NECA, CGS 21680C and CGS 24012. The cardiovascular pattern of responses to the two selective A2 agonists, CGS 21680C and CGS 24012, are distinct from those of NECA, the nonselective adenosine agonist. The responses to both CGS 21680C and CGS 24012 indicate that systemic vasodilation, with resultant cardioexcitation and stimulation of renin release, are the predominant hemodynamic effects of selective A2 agonists in the conscious SHR. In contrast, the cardiovascular effects produced by NECA are mediated by activation of both A1 and A2 receptors.

Hemodynamic effects of adenosine agonists in the conscious spontaneously hypertensive rat.

The present studies examined the underlying hemodynamic mechanisms contributing to the reduction in blood pressure observed in conscious spontaneously hypertensive rats after systemic administration of adenosine agonists. The effects produced by i.v. and i.a. injections of 2-phenylaminoadenosine [CV-1808, adenosine (A2) selective agonist], 5'-N-ethylcarboxamide adenosine (NECA, nonselective agonist), 2-chloroadenosine (2-CADO, A1 selective agonist) and cyclopentyladenosine (CPA, A1 selective agonist) were evaluated and compared to those of hydralazine. All agents produced hypotensive effects after bolus i.v. injection. Although CPA, NECA and 2-CADO elicited dose-dependent bradycardia, CV-1808 and hydralazine increased heart rate. These effects, with the exception of the hydralazine-evoked responses, were attenuated by prior treatment with 8-(p-sulfophenyl)theophylline (2 mg/kg/min), whereas both CV-1808 and hydralazine produced regional vasodilation, significant increases in blood flow occurred only after CV-1808 (3-30 micrograms/kg). The regional hemodynamic responses to NECA were more complex; low doses (0.1-1 microgram/kg) produced consistent reductions in regional vascular resistance, whereas at the highest dose renal vasoconstriction occurred. Although regional vasodilation occurred after 2-CADO, mesenteric vasoconstriction was observed subsequent to CPA administration. Whereas a significant increase in renin release was evident in animals treated with CV-1808 and hydralazine, no change occurred in response to the NECA-, 2-CADO- or CPA-induced hypotension. We conclude that the predominant hemodynamic response after selective activation of A2 receptors is one of regional vasodilation and hypotension leading to a reflex increase in heart rate and renin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae.

The dihydrofolate reductase gene (DFR1) from Saccharomyces cerevisiae has been mapped and sequenced. The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim. A 1.8-kb SalI-BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E. coli DHFR-deficient (folA) mutant. Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted Mr of 24230. The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources. The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage. The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control. Several potential 'TATA' boxes are located in the sequence 5' to the gene. Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast. We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.

Nifedipine gastrointestinal therapeutic system.

Convenient once-a-day dosage regimens are highly desirable in general, and especially for the treatment of asymptomatic diseases such as essential hypertension. Nifedipine is an insoluble, short-acting calcium channel blocker that presents a difficult technical challenge for formulation in a constant 24-hour delivery dosage form. Once-a-day dosage forms have been developed based on the gastrointestinal therapeutic system (GITS) push-pull osmotic pump configuration in three strengths with different drug delivery rates (mg/hour) per dose (mg), as 1.7/30, 3.4/60, and 5.1/90. The delivery rates of drug from these systems are controlled by their drug loading, composition of osmotic components, membrane properties, and dimensions. The release rates are independent of pH in the range from gastric pH = 1.2 to intestinal pH = 7.5. The release rates are independent of stirring rate and therefore unlikely to be influenced by motility in the gastrointestinal tract. The drug release rate from the nifedipine GITS dosage form in vivo in the gastrointestinal tract of dogs has been found to be equal to the release rate in vitro, indicating that the in vitro test is predictive of in vivo delivery. Following administration of the nifedipine GITS dosage forms to human subjects, absorption rates, calculated from resulting plasma concentrations, indicate that the cumulative amount of drug absorbed in humans over 24 hours is proportional to the amounts of drug delivered in vitro. Plasma concentrations are therefore predictable and remain relatively constant throughout the 24-hour dosing interval.

Central and peripheral inhibition of angiotensin converting enzyme (ACE) in the SHR: correlation with the antihypertensive activity of ACE inhibitors.

A series of five structurally distinct ACE inhibitors were evaluated for their ability to inhibit tissue ACE activity in the SHR after oral administration. In the first series of experiments, the ACE inhibitors captopril, enalapril, pentopril, CGS 14824A and CGS 16617 were given to groups of SHR at doses that produced a 15 to 20 mm Hg reduction in blood pressure within 1 hour. Under these conditions of dose and time, only captopril significantly inhibited brain ACE activity (43%), whereas inhibition of serum ACE activity ranged from 72% to 99% with these agents. Inhibition of aortic ACE activity ranged from 54% to 87%, and lung ACE inhibition varied from 50% to 83%. In the second series of experiments, SHR were administered higher doses of each ACE inhibitor such that these compounds produced peak reductions in blood pressure (-25 mm Hg to -33 mm Hg) within a range of 2 to 6 hours. When tissue ACE activity was measured at the time corresponding to peak reduction in blood pressure, all five ACE inhibitors produced a significant inhibition of brain ACE activity ranging from 21% to 76%. Serum ACE activity was almost completely inhibited by these agents, with the exception of captopril (62% inhibition). The inhibition of aortic ACE activity ranged from 79% to 99%, while the inhibition of lung ACE activity did not increase under these conditions. These data suggest that ACE inhibitors may exert their maximal antihypertensive effects by inhibiting ACE in vascular tissues and brain.

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

Genetic and biochemical consequences of thymidylate stress.

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.

Nuclear morphology of yeast under thymidylate starvation.

During early meiotic development the yeast Saccharomyces cerevisiae has a characteristic nuclear dense body (NDB). It is shown that the NDB can also be induced in vegetatively growing cells through the inhibition of thymidylate synthetase which causes depletion of the dTMP pool and arrests DNA synthesis. The observations on NDBs and recombination levels suggest that thymidylate-stressed cells may activate parts of the meiotic pathway and, conversely, cells on sporulation medium may sense, among other things, reduced thymidylate levels and respond to the several stimuli by entering the meiotic pathway.