Evidence that DNA damage is associated to phenylalanine blood levels in leukocytes from phenylketonuric patients

Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism, biochemically characterized by the accumulation of Phe and its metabolites in blood and tissues of affected patients. Treatment for PKU consists of a protein restricted diet supplemented with a mixture containing essential amino acids (other than Phe) and micronutrients. In recent years several authors have studied the pathomechanisms of the disease and demonstrated the existence of lipid and protein oxidative damage in PKU patients. In this work we investigated the in vivo and in vitro effects of Phe on DNA damage determined by the alkaline comet assay using silver staining and visual scoring. We found a dose-dependent effect of Phe on DNA damage in leukocytes from normal individuals incubated with different concentrations of Phe. Additionally, by analyzing blood leukocytes from two groups of treated PKU patients based on their blood Phe levels, we verified that the DNA damage index was significantly higher in PKU patients with high Phe blood levels (DI = 68.2 ± 12.3), compared to well-treated patients and the control group (healthy individuals). Furthermore, well-treated PKU patients had greater DNA damage (DI = 44.9 ± 7.6) relatively to controls (DI = 12.7 ± 4.1). Our present in vitro and in vivo findings indicate that DNA damage occurs in peripheral blood from PKU patients and is associated to Phe blood levels.     

Increased circulating IL-8 is associated with reduced IGF-1 and related to poor metabolic control in adolescents with type 1 diabetes mellitus

Background: A dysregulated growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis is well-recognized in children and adolescents with type 1 diabetes mellitus (T1DM). Decreased IGF-1 levels can also be found in chronic inflammatory diseases, while hyperglycemia promotes inflammatory cytokine production. Therefore, inflammatory cytokines may link poor metabolic control with GH/IGF-1 axis changes. This study examined the relationship between serum inflammatory cytokines and IGF-1 in adolescents (age 13–18) with TIDM in chronic poor (n = 17) or favorable (n = 19) glucose control. Poor control (PC) was defined as 3, consistent HbA1C > 9% during the previous 2 years, while favorable control (FC) was consistent levels of HbA1C < 9%. Results: HbA1C (FC: 7.5 ± 0.6%; PC: 10.5 ± 0.9%, p < 0.001) and interleukin (IL)-8 (FC: 3.7 ± 4.0 pg/ml; PC: 7.4 ± 4.3 pg/ml, p = 0.01) were increased and IGF-1 (FC: 536.5 ± 164.3 ng/ml; PC: 408.9 ± 157.1 ng/ml, p = 0.03) was decreased in patients with poor control compared to patients with favorable control. Moreover, IL-8 was inversely correlated with IGF-1 (r = −0.40, p = 0.03) and positively correlated with HbA1C (r = 0.36, p = 0.03). Conclusions: In adolescents with T1DM and chronic, poor glucose control, increased serum IL-8 is associated with reduced IGF-1 suggesting a pro-inflammatory milieu that may contribute to alterations in the GH/IGF-1 axis.     

The effect of the nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells

Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 ± 2 years, BMI 31.7 ± 1.2 kg/m², fasting plasma glucose 9.52 ± 0.80 mmol/L) and eight healthy individuals (aged 46 ± 2 years, BMI 27.1 ± 1.5 kg/m², fasting plasma glucose 4.69 ± 0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 μM) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was also examined at a concentration of 50 μM. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P = 0.03) but not the therapeutic SNP doses (P = 0.60) or SNAP (P = 0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.     

Evidence from genetic studies among rs2107538 variant in the CCL5 gene and Saudi patients diagnosed with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a chronic and metabolic disorder that affects the adult population. Chemokines are proinflammatory cytokines that play a role in the development of chronic diseases such as obesity, gestational diabetes, and T2DM. The C-C Motif Chemokine Ligand 5 (CCL5) gene plays a role in antiviral immunity, tumor development, obesity, impaired glucose tolerance, and T2DM. This study aimed to investigate the genetic role of the rs2107538 variant in the CCL5 gene in Saudi patients with T2DM. Sixty subjects with T2DM patients and 60 healthy controls participated in this prospective case-control study. Prior to Sanger sequencing, genomic DNA was extracted and amplified with Polymerase chain reaction (PCR), after which the PCR products were purified. The collected data were used to conduct various statistical analyses to determine the relationship between T2DM and control subjects. The findings of the current study revealed a positive association for most parameters between T2DM and control subjects (p < 0.05). The frequency of genotypes (p = 0.002, AA vs.GG: p = 0.008, GA + AA vs. GG: p = 0.0002) and alleles (A vs. G: p = 0.0007) revealed a strong risk association. Multiple logistic regression with individual effects revealed a link between SBP and HDLc levels (p = 0.03). In patients with T2DM, waist (p = 0.001), TG (p = 0.0007), and LDLc (p = 0.0004) levels were all associated with the ANOVA. Finally, the rs2107538 variant was linked to an increased risk of T2DM in the Saudi Population. The GA and AA genotypes were strongly connected to the T2DM subjects. In order to rule out disease-causing variants in the global population, future research should use a large sample size.     

Association of ARHGEF11 R1467H polymorphism with risk for type 2 diabetes mellitus and insulin resistance in Chinese population

The human Rho guanine nucleotide exchange factor 11 (ARHGEF11), located on chromosome 1q21, is an activator of Rho GTPases involved in G protein signaling pathway known to regulate insulin secretion and action. The aim of our study was to evaluate the relationship between the previously reported R1467H G/A variant in ARHGEF11 and risk of type 2 diabetes mellitus (T2DM) and insulin resistance as well as metabolic traits in a Chinese population. We genotyped R1467H G/A polymorphism in 311 patients with T2DM and 328 control subjects in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol and DNA sequencing methods. The genotype and allele distributions of the R1467H G/A polymorphism were significantly different between the T2DM group and the normal control group (P = 0.024, 0.018, respectively) and we also found that the A allele carriers (GA + AA genotype) had markedly higher risk of T2DM as compared with the wild-type GG genotype after adjustment for gender, age, and BMI (OR = 1.578, 95% CI 1.126–2.212, P = 0.008). Moreover, in the T2DM group the A allele carriers had higher FPG, FINS, and HOMA-IR than that of the GG homozygote (P = 0.015, 0.029, and 0.007, respectively). FPG and HOMA-IR levels were also significantly increased from A allele carriers to GG homozygote in the control group (P = 0.017, 0.012, respectively). Our investigation suggests that the R1467H polymorphism of ARHGEF11 gene may contribute to susceptibility to T2DM and insulin resistance in a Chinese population.     

Butyrylcholinesterase K variant and the APOE-ε4 allele work in synergy to increase the risk of coronary artery disease especially in diabetic patients

We have previously shown that butyrylcholinesterase-K (BCHE-K, G1615A/Ala539Thr) variant increases the risk of coronary artery disease (CAD). In addition, we have found that the presence of APOE-ε4 allele augments the risk of CAD in patients with type II diabetes mellitus (T2DM/CAD). Here we explored the concomitant presences of two alleles of the BCHE-K and APOE-ε4 in increasing the risk of CAD or diabetes in T2DM patients with or without CAD and CAD patients without T2DM. This case–control study comprised 631 subjects undergoing their first coronary angiography. They were matched and randomly assigned into four groups: type II diabetic patients with no sign of CAD (T2DM), type II diabetic patients with CAD/ND (T2DM/CAD), CAD patients with no sign of diabetes (CAD/ND), and healthy individuals (NCAD/ND). BCHE-K variant and APOE genotypes were detected by PCR-RFLP and serum lipid level was measured enzymatically. We found that BCHE-K and APOE-ε4 allele act synergistically to increase the risk of CAD in both T2DM, non-diabetic and total CAD (TCAD = T2DM/CAD + CAD/ND) individuals. The level of synergy 1.5 and 1.2 fold are higher in CAD patients (OR = 4.5; P = 0.011) with T2DM than the non-diabetic CAD patients (OR = 3.07; P = 0.024) and TCAD patients (OR = 3.74; P = 0.018), respectively. The CAD subjects with and without T2DM and TCAD patients carrying both APOE-ε4 allele and BCHE-K had significantly lower plasma HDL-C (P values = 0.008, 0.047, and 0.036, respectively) and higher plasma LDL-C (P values = 0.025, 0.048, and 0.04, respectively), than that of the control carriers both APOE-ε4 and BCHE-K. We have found that BCHE-K and APOE-ε4 allele not only act synergistically to increase the risk of CAD, particularly in T2DM subjects in population from western Iran, who have high levels of LDL-C and low levels of HDL-C, suggesting that a specific therapeutic intervention should be considered for these particular groups of patients.     

Glucagon-like peptide-1 protects mesenteric endothelium from injury during inflammation

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability (L_p) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L_p was measured. Second, after systemic LPS injection, L_p was measured after subsequent perfusion with GLP-1. Thirdly, L_p was measured after LPS injection and perfusion with GLP-1 + GLP-1 receptor antagonist. Lastly, L_p was measured after LPS injection and perfusion with GLP-1 + inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC) ± SEM. GLP-1 had no effect on L_p (AUC: baseline = 27 ± 1.4, GLP-1 = 1 ± 0.4, p = 0.08). LPS increased L_p two-fold (AUC: LPS = 54 ± 1.7, p < 0.0001). GLP-1 reduced the LPS increase in L_p by 75% (AUC: LPS + GLP-1 = 34 ± 1.5, p < 0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + antagonist = 46 ± 2.0, p < 0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS + GLP-1 + cAMP inhibitor = 46 ± 1.5, p < 0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS + GLP-1 + PKA inhibitor = 56 ± 1.5, p < 0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.     

Association of type 2 diabetes susceptibility genes (TCF7L2, SLC30A8, PCSK1 and PCSK2) and proinsulin conversion in a Chinese population

TCF7L2 and SLC30A8 have been found to be associated with type 2 diabetes mellitus (T2DM) as well as with impaired proinsulin processing recently, enzymes encoded by PCSK1 and PCSK2 are reported to play an important role in the process of proinsulin conversion. To investigate whether the single nucleotide polymorphisms (SNPs) of TCF7L2, SLC30A8, PCSK1 and PCSK2 were associated with T2DM as well as with proinsulin conversion in a Han Chinese population from Chongqing. A case–control study was performed in Han Chinese subjects with normal control (n = 152) and T2DM (n = 227), we genotyped rs7903146 and rs11196218 at TCF7L2, rs13266634 at SLC30A8, rs3811951 at PCSK1 and rs2021785 at PCSK2. Plasma levels of proinsulin were measured with an Enzyme Linked Immunosorbent Assay (ELISA). Genotype distribution and associations with T2DM and fasting levels of proinsulin and proinsulin/insulin ratios were analyzed. We confirmed the association of risk allele of rs2021785 at PCSK2 with type 2 diabetes also existed in Han Chinese population [OR = 1.4489 with 95% CI (1.0285, 2.0412), P = 0.0335]. Rs13266634 at SLC30A8 had a tendency to be associated with fasting plasma levels of proinsulin (P = 0.0639 in additive model). We did not find the significant association between other SNPs and T2DM or fasting levels of proinsulin or proinsulin/insulin ratios. Our results provide evidence that the association of PCSK2 and T2DM was also existed in Han Chinese population in Chongqing. We were underpowered to detect the association between other SNPs and T2DM or proinsulin conversion.     

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R² value = 0.949; P < 0.01; n = 16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n = 6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 °C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P = 0.001) in DNA damage, as soon as 4 h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6 h of incubation and revealed individual animal variation. Freshly collected and incubated (37 °C) rhinoceros (n = 3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.     

Effects of Acute Swimming Exercise on Some Elements in Rats

The objective of the present study is to explore the effects of acute swimming exercise on plasma levels of some elements in rats, immediately after the exercise, and 24 and 48 h later. The study included 40 adult male rats of Spraque Dawley species, which were equally allocated to four groups. Group 1: General Control Group; Group 2: Swimming Group, the group that was decapitated immediately after 30-min acute swimming exercise; Group 3: Swimming Group, the group that was decapitated 24 h after 30-min acute swimming exercise; Group 4: Swimming Group, the group that was decapitated 48 h after 30-min acute swimming exercise. Plasma copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P), selenium (Se), zinc (Zn) levels were determined according to atomic emission method in the blood samples collected from the animals by decapitation method. Measurements conducted immediately after acute swimming exercise (group 2) showed a significant decrease in Se and Zn levels (p < 0,01) and a significant increase in P levels (p < 0,01), when compared to group 1. Measurements carried out 24 h after the exercise (group 3) demonstrated a significant increase in all parameters except for Mg, in comparison to groups 1 and 2 (p < 0,01). It was seen in the measurements made 48 h after the exercise (group 4) that all parameters were restored to control values. The results of our study show that acute swimming exercise significantly changes plasma Cu, Fe, P, Se, and Zn levels.     

Mechanical modulation of atrial flutter cycle length

Although atrial flutter (AFL) is considered a highly regular rhythm, small fluctuations in cycle length have been described. The mechanisms responsible for these interval oscillations have been investigated by recent studies in humans which have shown that cyclic variations in atrial volume and pressure following ventricular contraction may account for the spontaneous variability of AFL. Other studies have shown that variations in the dimensions of the atria, caused by hemodynamical alterations due to imposed manoeuvres, directly modify the rate of AFL. All this evidence has led to the development of the mechano-electrical feedback (MEF) hypothesis, which assumes that changes in atrial volume directly affect AFL cycle length variability by modifying the conduction properties of the circulating impulse in the atrium.In the present study, we re-examined the variability pattern of typical AFL by spectral analysis aiming to support the MEF hypothesis for AFL cycle length variability. In a study population of 30 patients with typical AFL, we observed that AFL cycle length presented a spontaneous beat-to-beat variability, composed of two oscillations: a main oscillation at the frequency of ventricular contraction (1.70±0.48 Hz, spectral power: 15.4±17.6 ms²) and a second oscillation at the frequency of respiration (0.32±0.07 Hz, spectral power: 2.9±2.6 ms²). Both ventricular and respiratory oscillations persisted after pharmacologic autonomic blockade (ventricular spectral power: 17.7±14.7 ms² (before block) vs 20.2±18.3 ms² (after block), p=NS; respiratory spectral power: 6.0±3.8 ms² (before block) vs 5.0±3.4 ms² (after block), p=NS), suggesting a non-neurally mediated underlying mechanism. Contrary to respiratory modulation of heart rate during sinus rhythm, respiratory AFL cycle length oscillations displayed a reverse pattern, with longer cycle lengths during inspiration and shorter during expiration (AA_insp=223.2±28.6 ms vs AA_exp=221.1±28.2 ms, p<0.0005), which was consistent with a mechanical modulation of AFL reentry.The use of spectral analysis techniques applied to ventricular interval series and combined with computer simulations of atrioventricular conduction showed that the respiratory oscillation of atrial cycle length determined an oscillation in ventricular intervals with longer intervals during inspiration and shorter during expiration (VV_insp=639.9±186.0 ms vs VV_exp=634.8±182.9 ms, p<0.05). Ventricular interval oscillations resulted amplified by a factor 1.8 with respect to corresponding atrial cycle length oscillations. Thus, the mechanical fluctuations in AFL cycle length, although of small amplitude, might become clinically relevant through a magnified effect on ventricular variability.     

Oxidative stress and endogenous DNA damage in blood mononuclear cells may predict anti-SARS-CoV-2 antibody titers after vaccination in older adults

Immune senescence in the elderly has been associated with chronic oxidative stress and DNA damage accumulation. Herein we tested the hypothesis that increased endogenous DNA damage and oxidative stress in peripheral blood mononuclear cells of older adults associate with diminished humoral immune response to SARS-CoV-2 vaccination. Increased oxidative stress and DNA double-strand breaks (DSBs) were detected in 9 non-immunocompromised individuals aged 80–96 years compared to 11 adults aged 27–44 years, before, as well as on days 1 and 14 after the first dose, and on day 14 after the second dose of the BNT162B2-mRNA vaccine (all p < 0.05). SARS-CoV-2 vaccination induced a resolvable increase in oxidative stress and DNA damage, but individual DSB-repair efficiency was unaffected by vaccination irrespective of age, confirming vaccination safety. Individual titers of anti-Spike-Receptor Binding Domain (S-RBD)-IgG antibodies, and the neutralizing capacity of circulating anti-SARS-CoV-2 antibodies, measured on day 14 after the second dose in all participants, correlated inversely with the corresponding pre-vaccination endogenous oxidative stress and DSB levels (all p < 0.05). In particular, a strong inverse correlation of individual pre-vaccination DSB levels with both the respective anti-S-RBD-IgG antibodies titers (r = ?0.867) and neutralizing capacity of circulating anti-SARS-CoV-2 antibodies (r = ?0.983) among the 9 older adults was evident. These findings suggest that humoral responses to SARS-CoV-2 vaccination may be weaker when immune cells are under oxidative and/or genomic stress. Whether such measurements may serve as biomarkers of vaccine efficacy in older adults warrants further studies.     

Elevated endogenous testosterone concentrations potentiate muscle androgen receptor responses to resistance exercise

The purpose of this study was to determine the influence of endogenous circulating testosterone (T) on muscle androgen receptor (AR) responses to acute resistance exercise (RE). Six healthy men (26 ± 4 years; 176 ± 5 cm; 75.8 ± 11.4 kg) performed a knee extension exercise protocol on two occasions separated by 1–3 weeks. Rest preceded one trial (i.e., control [CON] trial) and a high-volume upper-body RE protocol designed to increase circulating T preceded the other trial (i.e., high T [HT] trial). Serial blood samples were obtained throughout each trial to determine circulating T concentrations. Biopsies of the vastus lateralis were obtained pre-RE (REST), 10-min post-RE (+10), and 180-min post-RE (+180) to determine muscle AR content. Circulating T concentrations remained stable during CON. Alternately, HT significantly (p ≤ 0.05) increased T concentrations above resting values (+16%). Testosterone area-under-the-time curve during HT exceeded CON by 14%. AR content remained stable from REST to +10 in both trials. Compared to the corresponding +10 value, muscle AR content at +180 tended to decrease during CON (−33%; p = 0.10) but remained stable during HT (+40%; p = 0.17). Muscle AR content at +180 during the HT trial exceeded the corresponding CON value. In conclusion, acute elevations in circulating T potentiated muscle AR content following RE.     

Asymmetrical myocardial expression of natriuretic peptides in pacing-induced heart failure

High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n = 8) and without (control, n = 6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65 ± 0.11 vs. 0.35 ± 0.04, p = 0.02), but not in the OS (BNP/GAPDH: 0.36 ± 0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089 ± 0.036, PS 0.289 ± 0.23, OS 0.54 ± 0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69 ± 0.13; PS = 1.56 ± 0.19; OS = 1.70 ± 0.42 pg/mg protein; p = 0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.     

Clicker increases resistance to extinction but does not decrease training time of a simple operant task in domestic dogs (Canis familiaris)

Despite its popularity among pet owners and professional trainers, we are not aware of any studies that have investigated the efficacy of clicker training in canines. To this end, we taught 35 basenjis to nose-touch an orange traffic cone. Upon meeting pre-determined criteria, dogs progressed through: (1) training trials, wherein correct responses were followed immediately with a click plus food (clicker group) or food alone (control group); (2) strengthening trials, wherein dogs received the same reinforcement protocol as in training trials, except nose-touching behaviour was variably reinforced; and (3) extinction trials, wherein food was withheld from both groups, but dogs in the clicker group received a click alone for nose-touches. We found that the clicker and control groups did not differ with regard to the number of trials or the time required to meet training or strengthening criteria (P > 0.05 for all). However, the clicker group required significantly more trials (log₁₀ transformed means ± S.E. = 1.6 ± 0.03 trials versus 1.4 ± 0.03 trials, P < 0.001) and more time (log₁₀ transformed means ± S.E. = 2.85 ± 0.03 s versus 2.73 ± 0.03 s, P = 0.008) to reach extinction criterion. Additionally, younger dogs required fewer training (, P = 0.001) and strengthening (, P = 0.029) trials and less training (, P = 0.005) and strengthening (, P = 0.013) time to meet criteria than did older dogs. However, no age effect was found on extinction for either the number or duration of trials (P > 0.05 for both), implying that persistence in previously reinforced behaviour did not influence the age sensitivity found in task acquisition. Overall, these results suggest that, whereas the clicker may prolong behaviour without primary reinforcement, it does not reduce the training time of a simple operant task in dogs when primary reinforcement is briefly delayed. We speculate that the clicker may be most useful in maintaining established behaviours when primary reinforcement is unavailable or when its delivery is impractical. Additionally, we found that basenji dogs may become progressively impaired with age in the acquisition of stimulus-reward contingencies.     

Inhibitory effect of aluminium on calcium absorption in small intestine of rats with different thyroid hormone status

To analyse the influence of thyroid status on the effect of aluminium (Al) upon intestinal calcium (Ca) absorption, adult male Wistar rats with experimentally altered thyroid hormones circulating levels, were orally treated (o.g.) with 0 (control), or 50 mg elemental Al (as chloride)/kg body weight (b.w.) per day, for a 14 d period. Hyper- and hypo-thyroid conditions were respectively achieved by means of administration of either sodium levothyroxine (50 μg/kg b.w. per day, o.g.) or methimazole, a thyroxine synthesis inhibitor (1 mg/kg b.w. per day, o.g.). In duodenum–jejunum segments, in vitro mucosa-to-serosa ⁴⁵Ca flux (JCa_ms) and kinetics of ⁴⁵Ca uptake in isolated enterocytes, were determined. In serum, concentrations of thyroxine (T4) and triiodothyronine (T3) were measured by chemiluminescent enzyme immunoassay. Unlike non-Al-treated rats, JCa_ms of Al-exposed rats decreased as serum levels of T4 and T3 increased, showing a significant inverse correlation in both cases (T4: r² = 0.414, P = 0.024; T3: r² = 0.443, P = 0.018). Enterocytes isolated from rats treated with Al plus thyroxine showed a reduction of both maximum Ca uptake (4.86 ± 0.44 vs. 6.85 ± 1.04 nmol Ca/mg protein, P < 0.05) and K_m (0.84 ± 0.18 vs. 1.05 ± 0.36 mM, P < 0.05) when compared to control. The observed variability in the Al effect on Ca transport with thyroid status of rats could be reflecting a negative interaction of Al with thyroid hormone action mechanisms on intestinal Ca absorption, which would take place mainly at Ca entry into enterocyte from lumen.     

Acute and delayed responses of C-reactive protein,malondialdehyde and antioxidant markers after resistance training session in elite weightlifters: Effect of time of day

The aim of this study was to investigate the effect of an Olympic-Weightlifting-session followed by 48-h recovery period on the oxidative and antioxidant parameters’ diurnal variation. Nine weightlifters (21?±?0.5 years) performed, in randomized order, three Olympic-Weightlifting-sessions at 08?h:00, 14?h:00 and 18?h:00. Blood samples were collected: at rest and 3?min and 48?h after each session. C-reactive protein (CRP), rate of lipid peroxidation and antioxidant activities were assessed. At rest, analysis of variance showed a significant time of day (TOD) effect (p?<?0.05) for uric acid, catalase and glutathione peroxidase with higher values at 14?h:00 and 18?h:00 compared with 08?h:00. However, no significant TOD effect for malondialdehyde, total bilirubin and CRP was observed. Given the profound changes (p?<?0.001) in the post-training session values, these diurnal variations have been altered immediately and even 48?h after the training sessions. Despite the significant decreases in the post-training values after the 48-h recovery period (p?<?0.05), levels of lipid peroxidation and enzymatic defense remained elevated (p?<?0.05) 48?h after the morning training session. However, after the afternoon and evening sessions, the same period was sufficient to return values to the baseline levels. In conclusion, the morning session seems to generate the most important acute and delayed lipid peroxidation responses. Therefore, weightlifting coaches should avoid scheduling their training sessions in the morning-hours.     

Association of a polymorphism of ABCB1 with obesity in Japanese individuals

The aim of the present study was to identify gene polymorphisms that confer susceptibility to obesity. A total of 5448 unrelated Japanese individuals from two independent populations were examined: subject panel A comprised 4252 individuals who visited participating hospitals; subject panel B comprised 1196 community-dwelling elderly individuals. The genotypes for 95 polymorphisms of 67 candidate genes were determined. The χ² test revealed that six polymorphisms were related (p < 0.05) to the prevalence of obesity in subject panel A; after application of Bonferroni's correction, however, only the 2677G → A/T polymorphism (rs2032582) of the ATP-binding cassette, subfamily B, member 1 gene (ABCB1) was significantly associated (p = 0.0003) with obesity. Subsequent multivariable logistic regression analysis also revealed that the 2677G → A/T polymorphism of ABCB1 was significantly associated with obesity. For validation of this association, the 2677G → A/T polymorphism of ABCB1 was examined in subject panel B and again found to be significantly associated with obesity. Body mass index was significantly (p = 0.01) greater for individuals with the variant T allele of this polymorphism than for those with the GG genotype in the combined subject panels A and B. Our results suggest that the ABCB1 genotype may prove informative for assessment of genetic risk for obesity in Japanese individuals.     

DNA damage in storage cells of anhydrobiotic tardigrades

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.