Age-related changes in acute central leptin effects on energy balance are promoted by obesity.

Leptin is a key catabolic regulator of food intake (FI) and energy expenditure. Both aging and obesity have been shown to induce leptin-resistance. The present study aimed to analyze age-related changes in the anorexigenic and hypermetabolic responsiveness to acute intracerebroventricular leptin administration in different age-groups of normally fed male Wistar rats (adult and old rats from 3 to 24months of age, NF3 to NF24, respectively). The expressions of the long form of the leptin receptor (Ob-Rb) and inhibitory SOCS3 genes were also assessed by quantitative RT-PCR in the arcuate nucleus (ARC). The influence of high-fat diet-induced obesity (HF) on the anorexigenic leptin effects were also tested in younger and older middle-aged groups (HF6 and HF12). Leptin-induced anorexia varied with age: leptin suppressed re-feeding FI (following 48-h fasting) strongly in young adult (NF3), but not in younger or older middle-aged (NF6 or NF12) or in aging (NF18) rats. However, anorexigenic leptin effects reached statistical significance again in old NF24 rats. Leptin-induced hypermetabolism, on the other hand, showed monotonous age-related decline and disappeared by old age. Ob-Rb expression declined until 12months of age followed by a partial recovery in NF18 and NF24 groups. On the other hand, SOCS3 expression was high in NF6 and NF18 and to some extent in NF24 rats. Age-related alterations of Ob-Rb and SOCS3 expression in the ARC may partly contribute to the explanation of age-related variations in anorexigenic but not hypermetabolic leptin effects. High-fat diet-induced obesity was associated with resistance to leptin-induced anorexia in HF6, similar to that seen in NF6. However, instead of the expected leptin-resistance in HF12, a strong leptin-induced suppression of re-feeding was detected in these obese middle-aged rats. Our results suggest that acute central effects of leptin on anorexia and hypermetabolism change in disparate ways during aging, implying separate mechanisms (e.g. signal transduction pathways) of different leptin actions. The age-related pattern shown by leptin-induced anorexia may contribute to the explanation of middle-aged obesity, and partly to that of aging anorexia. Our findings concerning obese rats are in accord with previous observations on anorexigenic effects of peripherally administered cholecystokinin: diet-induced obesity appeared to accelerate the development of age-related regulatory alterations. Similarly, our present data also raise the possibility that chronic diet-induced obesity promotes responsiveness to centrally applied leptin at least concerning anorexigenic effects.

Age-related alterations in the central thermoregulatory responsiveness to alpha-MSH.

Alpha-melanocyte-stimulating-hormone (alpha-MSH) is a neuropeptide that induces weight loss via its anorexigenic and hypermetabolic/hyperthermic effects. Two major public health problems of the human population involving energy balance (i.e. middle-aged obesity and aging cachexia) also appear in other mammals, therefore age-related regulatory alterations may also be assumed in the background. Previous studies demonstrated characteristic age-related shifts in the anorexigenic effects of centrally applied alpha-MSH with strong effects in young adult, diminished efficacy in middle-aged and very pronounced responsiveness in old rats. The present study aimed to investigate age-related changes in the acute central thermoregulatory responsiveness to an alpha-MSH injection in rats and to compare them with those of food intake-related responsiveness. Oxygen consumption (VO2), core (Tc) and tail skin temperatures (Ts, indicating heat loss) of male Wistar rats of different age groups (from 2 to 24 months of age), were recorded in an indirect calorimeter complemented by thermocouples upon intracerebroventricular alpha-MSH administration (0, 5 µg) at a slightly subthermoneutral environment (25-26 °C). Acute alpha-MSH-induced rises in VO2 and Tc were most pronounced in the young adult age-group. In these rats the hyperthemic effects were somewhat diminished by an activation of heat loss. Juvenile animals showed weaker hyperthermic responses, middle-aged rats none at all. Alpha-MSH-induced hyperthermia became significant again in old rats. Acute thermoregulatory (hypermetabolic/hyperthermic) responsiveness to alpha-MSH shows a distinct age-related pattern similar to that of acute anorexigenic responsiveness. Thus, our results may also contribute to the explanation of both middle-aged obesity and aging cachexia.

Association of the tumor necrosis factor -308 A/G promoter polymorphism with Tourette syndrome.

Several lines of evidence suggest that certain subtypes of obsessive-compulsive and tic disorders might be paediatric manifestations of post-streptococcal autoimmunity caused by cross-reactive autoantibodies. As tumor necrosis factor (TNF) is known to play a seminal role in coordinating the humoral immune response, TNF gene polymorphisms have been proposed as genetic risk factors both in obsessive-compulsive disorder (OCD) and Tourette syndrome (TS). The aim of this study was to investigate two TNF promoter polymorphisms (-238 A/G: rs361525 and -308 A/G: rs1800629) on the genetic susceptibility to OCD and TS in a child psychiatric sample (102 patients with OCD and 117 patients with TS). In the case-control set-up, the genotype and allele frequencies were compared to a control group from the general population (n = 405). As a control child psychiatric sample, 194 children with attention-deficit hyperactivity disorder were also genotyped. Our results revealed that the TNF -308 G-allele was more frequent in children with TS compared to controls (90.2% vs 84.8%, P = 0.037). For confirmation of this genetic association, a family-based analysis, the transmission disequilibrium test was used, which showed preferential transmission of the G-allele to patients with TS (nominal P-value 0.011). Moreover, this allele was also transmitted more frequently to children with tic symptoms (nominal P-value 0.039). No association was found between OCD or obsessive-compulsive symptoms and the studied TNF polymorphisms. Based on these findings, the TNF -308 G-allele can be associated with Tourette syndrome, highlighting the potential pathophysiological role of TNF dysregulation.

Age and nutritional state influence the effects of cholecystokinin on energy balance.

Cholecystokinin (CCK) is anorexic, irrespective whether it is applied intraperitoneally (IP) or intracerebroventricularly (ICV) in male Wistar rats. The metabolic effects depend on the route of administration: by the IP route it elicits hypothermia (presumably by type-1 receptors, CCK1R-s), while ICV administration is followed by fever-like hypermetabolism and hyperthermia via activation of CCK2R-s, which latter response seems to be most important in the postprandial (compensatory) hypermetabolism. The efficacy of the IP injected CCK varies with age: it causes strong anorexia in young adult 4 and 6-months old and again in old rats (aged 18-24 months), but the middle-aged (12-month old) ones seem to be resistant to this effect. Such pattern of effects may contribute to the explanation of age-related obesity observed in middle-aged animals as well as to the aging anorexia and loss of body weight in old ones. Diet-induced obesity accelerates the appearance of CCK-resistance as well as the return of high sensitivity to CCK in further aging, while chronic calorie-restriction prevents the development of resistance, as if the speed of the age-related regulatory changes was altered by the nutritional state. The effects of ICV applied CCK also change with age: the characteristic anorexic and hypermetabolic/hyperthermic effects can be observed in young adult rats, but the effects gradually and monotonically decline with age and disappear by the old age of 24 months. These disparate age-related patterns of CCK efficacy upon peripheral or central administration routes may indicate that although both peripheral and central CCKR-s exert anorexic effects, they may have dissimilar roles in the regulation of overall energy balance.

Anorexic effect of peripheral cholecystokinin (CCK) varies with age and body composition (short communication).

Obesity of middle-aged mammals is followed at old age by anorexia and cachexia leading to sarcopenia. Complex age- and body composition-related alterations in the regulation of energy homeostasis may be assumed in the background. We aimed to test the possible contribution of age- and body composition-related changes of satiety responses to catabolic brain-gut-axis peptide cholecystokinin (CCK) to these alterations in energy balance during aging. Male Wistar rats (6-8 animals/group) aged 2 months (juvenile), 3 months (young adult), 6 or 12 months (early or late middle-aged), and 24 months (old) were injected intraperitoneally with 5 µg CCK-8 prior to re-feeding after 48-h food-deprivation. CCK suppressed re-feeding in young adult (26.8%), early middle-aged (35.5%), and old (31.4%) animals, but not in juvenile or late middle-aged rats (one-way ANOVA). CCK-resistance of 12 months old rats was prevented by life-long calorie-restriction: CCK suppressed their re-feeding by 46.8%. Conversely, in highfat diet-induced obese 6 months old rats CCK failed to suppress re-feeding. In conclusion, age-related changes in satiety responsiveness to CCK may contribute to the age-related obesity of middle-aged as well as to the anorexia of old animals. CCK-responsiveness is also influenced by body composition: calorie-restriction prevents the resistance to CCK, pre-existing obesity enhances it.

Alterations in the peptidergic regulation of energy balance in the course of aging.

With advancing age most aspects of the peptidergic regulation of energy balance are altered. The alteration involves both the peripheral peptides derived from the adipose tissue or the gastrointestinal tract and the peptides of the central nervous system (brainstem and hypothalamus). In general, the expression of orexigenic peptides and their receptors decreases with age, while that of the anorexic ones rather increases, but not simultaneously and not in a linear fashion. Apart from such quantitative changes, the efficacy of the related peptides may also change with age. These changes are not necessarily linear, either: instead of continuous decline or increase of its effects, the effects of a peptide may become less pronounced in some phases of aging and much enhanced in other ones. Comparing the individual peptides, the phasic alterations in their anabolic or catabolic roles in the regulation of energy balance may exhibit dissimilar time-patterns. In addition, within the overall anabolic or catabolic effects, the feeding and metabolic actions of certain peptides may not change simultaneously. Altogether, as compared with young adults, in middle-aged animals or individuals the anabolic processes (increased food intake with decreased energy expenditure) seem to prevail, which processes may contribute to the explanation of age-related obesity, while in the old ones the catabolic processes (anorexia with enhanced metabolic rate) dominate, which possibly explain the aging anorexia, frailty and sarcopenia.

Central alpha-MSH infusion in rats: disparate anorexic vs. metabolic changes with aging.

UNLABELLED: Changes of the anorexigenic and hypermetabolic components of the overall catabolic effect of alpha-MSH were studied in rats as a function of age. In male Wistar rats a 7 day-long intracerebroventricular infusion of alpha-MSH suppressed food intake and caused a fall in body weight in 2 and 3-4 month-old (young) groups, but it was most effective in the 24 month-old group and had hardly any effect in the 12 month-old (middle-aged) animals. In contrast, metabolic rate as well as biotelemetric measurements of core temperature and heart rate revealed the most pronounced hypermetabolic effects of such infusions at age 12 months. The hypermetabolic effect was still high in the oldest group, but low in the younger groups. IN CONCLUSION: Changes of the anorexigenic and hypermetabolic effects in the course of aging are not concordant. The overall catabolic activity of alpha-MSH is smallest in the middle-aged and highest in the oldest group.

Genetic factors of reaction time performance: DRD4 7-repeat allele associated with slower responses.

Twin studies indicate substantial inherited components in cognitive abilities. One of the most extensively studied candidate genes of cognitive functioning is the dopamine D4 receptor gene (DRD4), which has been suggested to be related to attentional disorders. Based on reaction time data of 245 Caucasians participating in different cognitive tasks, slower responses characterized the group with the 7-repeat allele. This effect was present in both sexes and was not because of fatigue. To our knowledge, this is the first report on significant association (P = 0.0001) between the DRD4 variable number of tandem repeat (VNTR) polymorphism and response latencies in a non-clinical adult sample. Other studied dopaminergic polymorphisms did not show an association with reaction time. These results illustrate that speed-of-performance measures derived from multiple reaction time tasks using standardization procedures could be promising tools to detect unique genetic effects in the background of cognitive abilities.

Dopamine D4 receptor and serotonin transporter gene effects on the longitudinal development of infant temperament.

Existing studies of the effect on infant temperament of the 48 base pair variable number of tandem repeats polymorphism in exon 3 of the dopamine D4 receptor gene, DRD4 VNTR, and the serotonin transporter-linked polymorphic region, 5-HTTLPR, have provided contradictory results, and age seems to be an important factor. The present study investigated the effect of these two polymorphisms on the stability of infant temperament between 4 and 9 months of age. Furthermore, the effect of a recently discovered single nucleotide polymorphism which modulates the 5-HTTLPR (rs25531) was investigated in relation to infant temperament. The study sample consisted of 90 infants, who were assessed by parental report at the two ages under consideration using the Revised Infant Behavior Questionnaire. It was found that infants carrying the 7-repeat allele of the DRD4 VNTR had higher levels of Negative Affect. Furthermore, there was an interaction between DRD4 VNTR and 5-HTTLPR genotype such that infants with the DRD4 VNTR 7-repeat allele and the highest expressing 5-HTTLPR genotype (L(A) L(A) ) had the highest level of Negative Affect. These effects were largely driven by scores on the Falling Reactivity scale. Genetic effects were stable across age. The results emphasize the need for developmental studies of genetic effects on temperament.

Polymorphisms of beta defensins are associated with the risk of severe acute pancreatitis.

AIMS: Bacterial translocation from the intestinal tract plays an important role in severe acute pancreatitis (AP). Human ß-defensins are a family of antimicrobial peptides present at the mucosal surface. The aim of this study was to investigate the relevance of single nucleotide polymorphisms (SNPs) in the DEFB1 gene and copy number polymorphisms of the DEFB4 genes in AP. METHODS: 124 AP patients (30 with mild and 94 with severe disease) and 100 healthy controls were enrolled in the study. Three SNPs of the DEFB1 gene [G-20A (c.-20G→A), C-44G (c.-44C→G) and G-52A (c.-52G→A)] were genotyped by Custom TaqMan assay. The DEFB4 gene copy number was determined by means of a TaqMan real-time PCR assay. RESULTS: Significantly higher frequencies of the AA genotype of G-20A (c.-20G→A) and the AA genotype of G-52A (c.-52G→A) were observed among the patients with severe AP (SAP) compared with the healthy controls (38 vs. 20 and 41 vs. 18%, respectively). The GG protective genotype of C-44G (c.-44C→G) SNP was much less frequent (1%) among the patients than among the controls (9%). A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with SAP compared with the healthy controls (62 vs. 24%, respectively). CONCLUSIONS: The variations in the genes encoding human ß-defensin-1 and -2 may be associated with the risk of SAP. and IAP.

Age-dependence of alpha-MSH-induced anorexia.

Long-term regulation of energy balance involves two major trends: first age-related obesity develops in the middle-aged, later it is followed by anorexia of aging (sarcopenia and/or cachexia). A dynamic balance between orexigenic and anorexigenic neuropeptides is essential for the regulation of energy homeostasis. Special imbalances of neuropeptide effects may be assumed corresponding to different age-periods. Anorexia induced by acute alpha-MSH (alpha-melanocyte stimulating hormone; endogenous melanocortin agonist) injections was analyzed in male Wistar rats aged 6-9 weeks (juvenile), 3-4 months (young adult), 6 or 12 months (two middle-aged groups), 18 months (aging) and 24-26 months (old). Alpha-MSH injected through a preimplanted intracerebroventricular (ICV) cannula (compared with saline injection) dose-dependently suppressed spontaneous food intake and also re-feeding following 24-h fasting, but the rate of suppression varied between age-groups. An ICV injection of 5 microg alpha-MSH attenuated the 2-h re-feeding by 21.9+/-3.2% in juvenile rats, strongly (68.7+/-2.5%) suppressed it in young adults, the suppression became progressively weaker in the two middle-aged groups (55.7+/-4.9%, vs. 26.4+/-4.9%, respectively), but it turned extreme in aging (94.7+/-4.2%) and old (74.3+/-4.5%) rats. Body composition also changed with age: unlike the tibialis anterior muscle, the epididymal and retroperitoneal fat pads increased until middle-age and remained large even in old animals, while the measured indicator of muscle mass decreased in the oldest group. The food intake suppressing and body weight decreasing effects of a 7-day-long ICV infusion of 1 microg/h alpha-MSH were weakest in the 12-month-old and most pronounced in the 24 month-old rats. In conclusion, responsiveness to the anorexic effect of alpha-MSH varies with age, with a nadir of the curve in the middle-aged, and a peak in the aging and old animals. This age-related nadir of melanocortin-responsiveness may promote obesity in middle-aged rats, while the tendency for anorexia and incipient sarcopenia of old (still obese) rats may result from age-related melanocortin-hypersensitivity rather than from adiposity.

Suppression of food intake by intracerebroventricular injection of alpha-MSH varies with age in rats.

During the aging process of mammals first a phase of obesity and increased adiposity is observed in middle-aged subjects, then anorexia and loss of body weight (sarcopenia) at old age. A possible age-dependence of the anorexigenic alpha-melanocyte-stimulating-hormone (alpha-MSH) in these regulatory changes was studied. Male Wistar rats aged 6-8 weeks (juvenile), 3-4 months (young adult), 6 and 12 months (middle-aged), and 24-26 months (old) were equipped with chronic cannula to the lateral cerebral ventricle. The effect of 5 microg alpha-MSH injected through the cannula was analyzed on food intake evoked by 24-h food deprivation. Juvenile rats seemed almost resistant to alpha-MSH (21.9% suppression). In young adults alpha-MSH suppressed food intake by 68.7%. However, the alpha-MSH-induced anorexia was significantly less pronounced in middle-aged (55.7% or 26.4% in rats aged 6 or 12 months, respectively), and much more pronounced (73.3%) in old rats. The adiposity (judged by the relative amount of perirenal fat) increased until middle-age, but did not change between middle-age and old-age. It is concluded that changes in alpha-MSH responsiveness possibly contribute to both the age-related obesity in middle-aged rats and to the anorexia of old ones: first the adiposity then the age may be the important factor.

Molecular and behavioral analysis of the intron 2 repeat polymorphism in the canine dopamine D4 receptor gene.

Genetic polymorphisms in the human dopamine D4 receptor (DRD4) gene, especially the exon 3 variable number of tandem repeats (VNTR), have been related to several psychiatric disorders and personality traits. A homologous exon 3 VNTR has been described in dogs, and we previously showed an association between the DRD4 exon 3 polymorphism and activity/impulsivity trait in German shepherds. In this study, we present a detailed analysis of the intron 2 VNTR of the DRD4 gene. A short and a long form of the intronic variation were identified in 678 unrelated dogs from five breeds and in 22 wolves. For molecular analysis, the intron 2 region was cloned into a promoterless luciferase reporter vector that led to an elevation in transcriptional activity. Moreover, an allelic difference in promoter activity was detected, and a repressive effect of the long allele was observed. Behavioral analysis of 96 unrelated German shepherds showed a significant association between the social impulsivity endophenotype of the Greeting Test and both the exonic (P = 0.002) and the intronic (P = 0.003) VNTRs of the DRD4 gene. Moreover, an additive effect of the two polymorphisms was also shown (Spearman's rho = 0.356, P = 0.0004). In conclusion, these results give further support to our previous findings that the DRD4 gene is associated with dog behavior. We also present molecular evidence for the functional role of the intron 2 VNTR in the canine DRD4 gene.

Kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma.

UNLABELLED: A number of human diseases and pathological conditions were found to be associated with increased oxidative stress. In the literature several techniques are available for the assessment of oxidative stress, but most of them are not applicable for a routine medical laboratory due to the complex methodology and/or financial reasons. We report here on a simple, inexpensive, kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma. METHODS: This study involved 70 patients (47M/23F; mean age: 64.6 y; range: 16-85) admitted to our Department with a wide range of cardiovascular and peripheral vascular diseases. Three critically ill patients were assigned for monitoring purposes. Plasma AOPP were simultaneously determined using an end-point assay as reference method and by a kinetic method developed in our laboratory. Plasma fibrinogen concentration was measured according to the Clauss method. RESULTS: There was a highly significant correlation (r2 = 0.588; p < 0.0001) between AOPP concentration (reference method) and AOPP reactivity (kinetic method). Both AOPP concentration and AOPP reactivity also significantly correlated with plasma fibrinogen concentration (r2 = 0.780; p < 0.0001; r2 = 0.564; p < 0.0001). The three representative cases presented appear to support the relevance of our novel method in the monitoring of critically ill patients. CONCLUSIONS: This simple and inexpensive kinetic assay can be widely used in any routine laboratory interested in oxidative stress research. It is especially recommended for monitoring critically ill or other patients.

A comparison of two methods for the evaluation of the daily urinary fluoride excretion in Romanian pre-school children.

OBJECTIVE: To compare two different methods of estimating daily fluoride urinary excretion in pre-school children under stable fluoride intake conditions. DESIGN: Thirty-five healthy kindergarten children, permanent residents of Targu-Mures, Romania, where the average drinking water fluoride concentration is 0.12 mg F/L, participated on two separate occasions, when they were aged 4-6 and 5-7 years, respectively in the collection of a) a mid-morning spot urine sample and b) a 16-h time-controlled urine sampling. In case a), the ratio of concentrations of fluoride and creatinine were measured, while in case b) the rates of fluoride excretion in two separate 8-hour periods were used to estimate the 24-hour fluoride urinary excretion. RESULTS: The estimated average daily fluoride urinary excretion values (S.D.) were 0.318 (0.182) mg F/day for method a) and 0.341 (0.193) mg F/day for method b). These values were not significantly different (Mann-Whitney U test; p = 0.49). The estimated daily fluoride doses were 0.040 (0.021) and 0.043 (0.022) mg F/kg body weight/day, respectively. The latter values were not significantly different (Mann Whitney U test; p = 0.38). CONCLUSIONS: Results obtained suggest that under stable F-intake conditions the estimation of the daily fluoride urinary excretion by means of a mid-morning spot urine sample yields comparable results to those obtained with the more involved method of separate, two 8 h (16 h) time-controlled urine sampling recommended by the WHO. Use of spot urine sampling appears to be particularly useful for epidemiological studies.

Adenoviral vector transduction of the human deoxycytidine kinase gene enhances the cytotoxic and radiosensitizing effect of gemcitabine on experimental gliomas.

The aim of this work was to improve the cytotoxic and radiosensitizing effects of gemcitabine using a gene-directed enzyme prodrug therapy approach. Murine Gl261, rat C6 and human U373 glioma cell lines were transduced with an adenoviral vector encoding the human deoxycytidine kinase gene (Ad-HudCK). Intracranial tumors were established in C57BL/6 mice and Wistar rats using either wild-type or Ad-HudCK-transduced Gl261 and C6 glioma cells. In vitro growing cells and established tumors were treated with gemcitabine and irradiation either alone or in combination. Deoxycytidine kinase overexpression substantially increased both the toxic and radiosensitizing effects of gemcitabine in each cell line, but the enhancement rate varied: it was mild in the Gl261 cells and much stronger in the C6 and U373 cells. In vivo experiments showed a mild radiosensitizing effect of dCK overexpression both in the Gl261 and C6 models. The combination of dCK overexpression, gemcitabine treatment and irradiation improved the survival rate of C6 bearing rats significantly. In conclusion, overexpression of the dCK gene can improve the cytotoxic and radiosensitizing effect of gemcitabine both in vitro and in vivo in a tumor-specific manner.

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs.

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene (DRD4) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a) were detected in this breed, and genotype frequencies were in Hardy-Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele (P = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.

Smoking and a complement gene polymorphism interact in promoting cardiovascular disease morbidity and mortality.

We have demonstrated previously that carriers of a genotype called C4B*Q0 (silent allele of the C4B gene) have a substantially increased risk to suffer from myocardial infarction or stroke, and are selected out from the healthy elderly population. Because smoking carries a major risk for cardiovascular disease (CVD), it seemed worthwhile to study if these two factors interact. Study 1 involved 74 patients with angina pectoris (AP), 85 patients with recent acute myocardial infarction (AMI) and 112 survivors of a previous AMI and 382 controls from Iceland. Study 2 involved 233 patients with severe CVD and 274 controls from Hungary. Smoking habits were registered for each subject. The number of C4A and C4B genes was determined by phenotyping or genotyping. Compared to controls, C4B*Q0 carrier frequency was significantly higher at diagnosis in Icelandic smokers with AP (P = 0.005) and AMI (P = 0.0003) and Hungarian smokers with severe coronary artery disease (P = 0.023), while no such difference was observed in non-smoking subjects. Age-associated decrease in C4B*Q0 observed previously in two remote Caucasian populations was found, in the present study, to be associated strongly with smoking, and to already occur in smokers after age 50 years both in Iceland and Hungary. Our findings indicate that the C4B*Q0 genotype can be considered as a major covariate of smoking in precipitating the risk for AMI and associated deaths.

Deoxycytidine kinase is reversibly phosphorylated in normal human lymphocytes.

The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions.

Effects of intracellular calcium chelation and pifithrin-alpha on deoxynucleotide metabolism in human lymphocytes.

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-alpha, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-alpha-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.