Pyridoxamine prevents age-related aortic stiffening and vascular resistance in association with reduced collagen glycation

An increase in oxidative chemical modifications of tissue proteins by advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) has been implicated in normal aging. Pyridoxamine (PM), one of the three natural forms of vitamin B₆, has been identified as an inhibitor of AGE/ALE products formed during the autoxidation of carbohydrates and peroxidation of lipids. The current study seeks to determine whether PM intervention could prevent the age-related aortic stiffening and vascular resistance through its ability to inhibit the pathogenic cross-linking of glycated collagen. Male Fisher 344 rats at 15 months were treated daily with PM (1 g l^− 1 in drinking water) for 5 months and compared with the age-matched, untreated controls at 20 months. Pulsatile aortic pressure and flow signals were measured to perform the vascular impedance analysis. The anti-AGE antibody 6D12 was used to detect glycation-derived modification of aortic collagen, using protein blotting analysis. PM therapy attenuated the age-related increase in total peripheral resistance. An increase in wave transit time and aortic compliance by PM indicated that the drug improved aortic distensibility of the aged vasculature. This paralleled its reduction of AGE-collagen cross-links on aortas. Treatment of the old animals with PM also prevented the age-induced augmentation in vascular load imposed on the heart, as evidenced by an increased wave transit time and a decreased wave reflection factor. These findings suggest a partial role of PM in improving arterial mechanics by targeting the pathogenic formation of AGE-induced aortic collagen cross-links in old rats.     

F-fluorodeoxyglucose uptake in brown adipose tissue during insulin-induced hypoglycemia and mild cold exposure in non-diabetic adults

Objective

Hypoglycemia is associated with increased heat production and, despite of this, hypothermia. Heat production is likely to be mediated by sympathetic innervation. Brown adipose tissue is activated by cold exposure and stimulated by the sympathetic nervous system. We therefore examined the effect of hypoglycemia on uptake of the labeled glucose analogue ¹⁸ F-fluorodeoxyglucose in brown adipose tissue using positron emission tomography and computer tomography.

Methods

In nine healthy adults ¹⁸ F-fluorodeoxyglucose uptake as measure of brown adipose tissue activity was assessed in a cold environment (17 °C) during euglycemia (blood glucose 4.5 mmol/L) and hypoglycemia (2.5 mmol/L) using a hyperinsulinemic glucose clamp.

Results

Brown adipose tissue activity was observed in all participants. No difference was observed in the median (range) maximal standardized uptake values of ¹⁸ F-fluorodeoxyglucose in brown adipose tissue between euglycemia and hypoglycemia: 4.2 (1.0–7.7) versus 3.1 (2.2–12.5) g/mL (p = 0.7). Similarly there were no differences in mean standardized ¹⁸ F-fluorodeoxyglucose uptake values or total brown adipose tissue volume between euglycemia and hypoglycemia. Body temperature dropped by 0.6 °C from baseline during the hypoglycemic condition and remained unchanged during the euglycemic condition. There was no correlation between the maximal standardized uptake values of ¹⁸ F-fluorodeoxyglucose in brown adipose tissue and levels of counterregulatory hormones.

Conclusions

This study shows that there is a similar amount of ¹⁸ F-fluorodeoxyglucose uptake in brown adipose tissue during hypoglycemia when compared to euglycemia, which makes a role for systemic catecholamines in brown adipose tissue activation and a role for brown adipose tissue thermogenesis in hypoglycemia associated hypothermia unlikely. Future studies in humans should determine whether hypoglycemia indeed increases energy expenditure, and if so which alternative source can explain this increase.     

LCT-13910C > T polymorphism-associated lactose malabsorption and risk for colorectal cancer in Italy

Background

The activity of epithelial lactase (LCT) associates with a polymorphism 13910 bp upstream the LCT-encoding gene (LCT-13910C > T). The relationship between LCT-13910C > T polymorphism and risk for colorectal cancer is unclear.

Aims

We examined the relationship between the LCT-13910C > T polymorphism causing lactose intolerance and risk for colorectal cancer/polyps onset in the Italian population.

Patients and methods

793 subjects (306 with colorectal cancer, 176 with polyps and 311 controls) were genotyped for the LCT-13910C > T variant by TaqMan real time-PCR.

Results

Lactose malabsorption linked to the CC genotype did not associate with an increased risk for either colorectal cancer (OR = 1.041; 95% CI = 0.751–1.442; p = 0.868) or polyps (OR = 0.927; 95% CI = 0.630–1.363; p = 0.769). There was no association with colorectal cancer/polyps site. 60% of the subjects overall bore the CC genotype.

Conclusion

In the Italian population the LCT-13910C > T polymorphism is not associated to the risk for colorectal cancer or polyps.     

Early return of augmented wave reflection impairs left ventricular relaxation in aged Fisher 344 rats

Left ventricular (LV) relaxation is influenced by vascular loads imposed on the heart. The current study investigated the influence of the timing and magnitude of arterial wave reflection on LV isovolumic pressure relaxation, with a specific focus on the aging process. Fisher 344 rats aged 6, 18, and 24months were anesthetized and thoracotomized. Arterial wave reflection was characterized by wave transit time (τ(w)) and wave reflection factor (R(f)) using the impulse response of the filtered aortic input impedance spectra. Indices of LV pressure relaxation included peak -dP(LV)/dt and the isovolumic relaxation time constant (τ(e)). The vascular dynamic condition in the rats was characterized by (1) a progressive increase in R(f) and decrease in τ(w) associated with age, especially at 24months; and (2) a decline in aortic compliance (C(m)). Changes in LV relaxation consisted of a fall in peak -dP(LV)/dt and a rise in LV τ(e) with age. Taking LV τ(e) as the dependent variable and arterial R(f) and τ(w) as the two independent variables, multiple linear regression was employed to fit the data. The correlation among the three parameters reached significance (τ(e) =11.885+5.350×R(f)-0.213×τ(w); r=0.5823, p<0.05). This finding indicated that as arterial τ(w) shortened and arterial R(f) was augmented with age, LV τ(e) became more prolonged and late pressure relaxation slowed. Thus, the heavy reflection intensity with early return of the pulse wave reflection might account for the age-related deterioration in LV isovolumic pressure decay.     

Conversion of a yeast prion protein to an infectious form in bacteria

Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [PSI⁺] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology.     

Methyltransferase Set7/9 regulates p53 activity by interacting with Sirtuin 1 (SIRT1)

Numerous studies indicate that Sirtuin 1 (SIRT1), a mammalian nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylase (HDAC), plays a crucial role in p53-mediated stress responses by deacetylating p53. Nevertheless, the acetylation levels of p53 are dramatically increased upon DNA damage, and it is not well understood how the SIRT1-p53 interaction is regulated during the stress responses. Here, we identified Set7/9 as a unique regulator of SIRT1. SIRT1 interacts with Set7/9 both in vitro and in vivo. In response to DNA damage in human cells, the interaction between Set7/9 and SIRT1 is significantly enhanced and coincident with an increase in p53 acetylation levels. Importantly, the interaction of SIRT1 and p53 is strongly suppressed in the presence of Set7/9. Consequently, SIRT1-mediated deacetylation of p53 is abrogated by Set7/9, and p53-mediated transactivation is increased during the DNA damage response. Of note, whereas SIRT1 can be methylated at multiple sites within its N terminus by Set7/9, a methylation-defective mutant of SIRT1 still retains its ability to inhibit p53 activity. Taken together, our results reveal that Set7/9 is a critical regulator of the SIRT1-p53 interaction and suggest that Set7/9 can modulate p53 function indirectly in addition to acting through a methylation-dependent mechanism.     

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background

The number of CD34⁺ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34⁺ cells to the bone marrow.

Design and Methods

We analyzed genetic characteristics associated with CD34⁺ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days).

Results

Genetic variants in VCAM1 and in CD44 were associated with the number of CD34⁺ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34⁺ cells ×10⁶/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34⁺ cells ×10⁶ (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34⁺ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34⁺ cells ×10⁶/kg of donor and total CD34⁺ cells ×10⁶ (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively).

Conclusions

In conclusion, genetic variability in molecules involved in migration and homing of CD34⁺ cells influences the degree of mobilization of these cells.     

Germline promoter hypermethylation of tumor suppressor genes in gastric cancer

AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16^INK4a in suspected cases of hereditary gastric cancer (GC).METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1, CDH1 and P16^INK4a tumor suppressor genes. Genomic DNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfite DNA sequencing for a specific region of the MLH1 promoter. The methylation status of CDH1 or P16^INK4a was assayed using methylation-specific PCR. Clonal bisulfite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions.RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16^INK4a genes was detected.CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16^INK4a gene is not a frequent event.     

Reduced autophagy in livers of fasted,fat-depleted,ghrelin-deficient mice: Reversal by growth hormone

Plasma growth hormone (GH) and hepatic autophagy each have been reported to protect against hypoglycemia in the fasted state, but previous data have not linked the two. Here we demonstrate a connection using a mouse model of fasting in a fat-depleted state. Mice were subjected to 1 wk of 60% calorie restriction, causing them to lose nearly all body fat. They were then fasted for 23 h. During fasting, WT mice developed massive increases in plasma GH and a concomitant increase in hepatic autophagy, allowing them to maintain viable levels of blood glucose. In contrast, lethal hypoglycemia occurred in mice deficient in the GH secretagogue ghrelin as a result of knockout of the gene encoding ghrelin O-acyltransferase (GOAT), which catalyzes a required acylation of the peptide. Fasting fat-depleted Goat^−/− mice showed a blunted increase in GH and a marked decrease in hepatic autophagy. Restoration of GH by infusion during the week of calorie restriction maintained autophagy in the Goat^−/− mice and prevented lethal hypoglycemia. Acute injections of GH after 7 d of calorie restriction also restored hepatic autophagy, but failed to increase blood glucose, perhaps owing to ATP deficiency in the liver. These data indicate that GH stimulation of autophagy is necessary over the long term, but not sufficient over the short term to maintain blood glucose levels in fasted, fat-depleted mice.When animals undergo a complete fast, adaptive mechanisms maintain the supply of energy to vital organs. The classic metabolic response to an acute fast was delineated in humans more than 50 y ago, largely through the work of George Cahill and colleagues (1, 2). The initial response to fasting is glycogenolysis, which releases glucose from liver and muscle. At the same time, the liver begins to synthesize and release glucose de novo, initially using lactate, which is returned to the liver from muscle through the Cori cycle. Shortly thereafter, lipolysis is activated in adipose tissue, releasing fatty acids for combustion in muscle and for the supply of energy to fuel gluconeogenesis in the liver. Hepatic fatty acid oxidation also produces the ketone bodies acetoacetate and beta-hydroxybutyrate, which can partially replace glucose for energy in brain and muscle. This entire process is orchestrated by hormones, primarily through a drop in insulin and increases in glucagon, which activates gluconeogenesis in liver (2), and growth hormone (GH), which stimulates lipolysis in adipose tissue (3).In recent years, another source of gluconeogenic substrates in liver has been identified, namely autophagy (4, 5). Autophagy is the process by which intracellular double-membrane vesicles called autophagosomes ingest cytosolic proteins and organelles (6). Autophagosomes fuse with lysosomes, creating single-membrane bounded autolysosomes that expose the ingested contents to hydrolases that break down the ingested macromolecules. Fasting induces autophagy in liver (4). Some of the autophagic end products are combusted to produce energy, and some are used as substrates for gluconeogenesis. When autophagy is prevented through germline deletion of a required protein, animals develop profound hypoglycemia on fasting (4, 7, 8).Previous studies of fasting have been conducted in humans or animals that maintain adequate adipose tissue, allowing a continuous supply of fatty acids to liver and muscle. We know little about the maintenance of blood glucose when adipose triglycerides are exhausted and fatty acids are not available. Such a fat-depleted condition occurs in humans subjected to prolonged calorie deprivation through famine, anorexia, or other causes of cachexia (9, 10).Recently, our laboratory created an experimental model of calorie restriction in fat-depleted C57BL/6 mice (11–13). In this protocol, mice are fed daily with 40% of the chow diet that the mouse would normally consume. We call this 60% calorie restriction. The mice are fed each day at 6:00 PM. Within 1 d, they become ravenously hungry, and consume all of their food by 7:00 PM. They are then fasted for the next 23 h until they are fed once again. This cycle is repeated each day for 7–9 d. We measure blood glucose each day at 5:30 PM, after the 23-h fast and immediately before the next feeding.Over the first 4 d of this regimen, WT mice lose ∼30% of their body weight, which then stabilizes. By day 4, their body fat, as measured by NMR, constitutes <2% of body weight (11, 13). By day 5 or 6, fasting blood glucose values at 5:30 PM fall to 40–60 mg/dL, and are maintained at that level through day 8. Despite this moderate hypoglycemia, WT mice are alert and active. They have very low concentrations of free fatty acids in plasma (≤0.07 mM) and ketone bodies (≤0.12 mM). The hormonal response includes a marked decrease in insulin, increase in glucagon, and increases in ghrelin and GH, with the latter two rising progressively through 8 d (11, 13).Survival of these fasted, fat-depleted WT mice requires ghrelin, which acts by stimulating secretion of GH (11). Ghrelin, a 28-aa peptide produced primarily by enteroendocrine cells in the stomach and duodenum, acts in the arcuate nucleus of the hypothalamus and in somatotrophs in the pituitary to release GH (14–16). Ghrelin activity requires ghrelin O-acyltransferase (GOAT), which attaches octanoate, an eight-carbon fatty acid, to serine-3 of ghrelin before secretion (17, 18). Mice with germline deletions of ghrelin or Goat are unable to maintain viable levels of blood glucose when subjected to 60% calorie restriction as described above (11, 13). The calorie-restricted, ghrelin-deficient mice lose body weight and body fat as rapidly as WT mice, and become just as hungry, consuming all of their food within 60 min. The ghrelin-deficient mice are unable to stabilize their blood glucose. By day 7–8, their fasting blood glucose falls below 20 mg/dL. It rises promptly after each meal, only to fall progressively during the next 23-h fast. Profound hypoglycemia can be prevented by chronic infusion of either ghrelin or GH, begun before the onset of calorie restriction and maintained throughout (11, 13).Tracer experiments demonstrate that hypoglycemia in ghrelin-deficient, fat-depleted mice is caused by reduced production of glucose rather than by enhanced clearance (13). Plasma lactate, the major substrate for gluconeogenesis, falls in parallel with blood glucose (13). On day 7 of calorie restriction, blood glucose can be restored to WT levels by injections of pyruvate, lactate, or alanine, which provide energy and substrates for gluconeogenesis. Blood glucose is also restored by octanoate, a fatty acid that cannot be converted to glucose but can be oxidized to provide energy (13).Inasmuch as both hepatic autophagy and plasma GH are required to maintain blood glucose during fasting, the present study was designed to test the hypothesis that GH stimulates hepatic autophagy in starved, fat-depleted mice. The results demonstrate that hepatic autophagy is diminished in ghrelin-deficient mice and can be restored acutely by GH infusion. Acute restoration of hepatic autophagy does not prevent hypoglycemia, however. These results raise the possibility that GH-stimulated hepatic autophagy is necessary, but not sufficient, to maintain blood glucose in fasted, fat-depleted mice.     

A G-protein γ subunit mimic is a general antagonist of prion propagation in Saccharomyces cerevisiae

The Gpg1 protein is a Gγ subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI⁺], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI⁺] but also [PIN⁺], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI⁺] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted α-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Δ and gpb1Δ strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.     

Extensive heterozygosity in flanking microsatellites of Plasmodium falciparum Na/H exchanger (pfnhe-1) gene among Indian isolates

Plasmodium falciparum Na⁺/H⁺ exchanger-1 (pfnhe-1) gene has been proposed to be a possible marker for quinine resistance. Here, we describe the sequence analysis of the flanking microsatellites of the pfnhe-1 gene among 108 Indian P.falciparum isolates. Among the parasite population, a high degree of polymorphism was observed at all the 10 microsatellite loci within ±40 kb region of the pfnhe-1 gene where the number of alleles varied from 2 to 16 with a high expected heterozygosity ranging from 0.43 to 0.91 at these loci. Also, higher levels of heterozygosity have been observed in P.falciparum isolates collected from both low and high transmission and drug resistant areas. Furthermore, there was no association between QN resistance associated DNNND repeats in PFNHE-1 and the flanking microsatellite haplotypes. In conclusion, the observed high level of microsatellite polymorphism and absence of selective sweep in the flanking ±40 kb region of the pfnhe-1 gene could be an indication that there is no strong selection pressure on this target gene.     

Prognostic value of FGFR1 gene copy number in patients with non-small cell lung cancer: a meta-analysis

Background

A number of studies have investigated the relationship between fibroblast growth factor receptor1 (FGFR1) gene copy number and survival in non-small cell lung cancer (NSCLC) patients. However, conclusions reported by different parties seem to be inconsistent, especially regarding the differences among different histopathologic subtypes. To derive a more precise estimate of the prognostic significance of FGFR1 gene copy number, we have reviewed published studies and carried out a meta-analysis.

Methods

The meta-analysis was conducted in accordance with PRISMA guidelines. The required data for estimation of individual hazard ratios (HRs) for survival were extracted from the publications and an overall HR was calculated.

Results

We identified 6 eligible studies, all dealing with NSCLC. The global quality score ranged 32.5-80%, with a median of 53.33%. For FGFR1 amplification in three studies including differed according to histological type, the overall RR was 0.86 which 95% confidence interval (CI) was 0.75 to 0.99 and P value was 0.048. Combined HR for the six evaluable studies was 1.17 (95% CI: 0.95 to 1.43). In the subgroup of squamous cell lung cancer (SQCC), the combined HR was 1.24 (95% CI: 0.89 to 1.73). For the Asian populations’ studies, the combined HR was 1.67 (95% CI: 1.1 to 2.52).

Conclusions

FGFR1 amplification significantly was more frequent in SQCC. FGFR1 was not associated with poorer survival in patients with NSCLC. Furthermore studies will be needed in terms of survival implications.     

Influence of Xmn 1Gγ (HBG2 c.-211 C → T) Globin Gene Polymorphism on Phenotype of Thalassemia Patients of North India

Beta (β) thalassemia is the most common single gene disorder in India. It has been reported that in patients with β-thalassemia in the presence of Xmn 1^Gγ polymorphic site the level of fetal hemoglobin (HbF) is increased thereby reducing the severity of disease. To determine the prevalence of Xmn 1^Gγ polymorphic site and its effect on the clinical phenotype and HbF level in 39 β-thalassemia major and 62 thalassemia intermedia patients, along with response to hydroxyurea therapy in thalassemia intermedia cases. Status of Xmn 1^Gγ polymorphism was determined by polymerase chain reaction-restricted fragment length polymorphism procedure. The HbF level was determined using high performance liquid chromatography. Genotypes and allele frequencies of the Xmn 1^Gγ polymorphism did not vary significantly between the various thalassemia groups. HbF levels were observed to be significantly increased and age at presentation was significantly greater in presence of Xmn 1^Gγ polymorphic site on both alleles as compared to its absence in thalassemia major but not in thalassemia intermedia cases. The response of hydroxyurea in thalassemia intermedia was found only in a few patients irrespective of their Xmn 1^Gγ status. Xmn 1^Gγ polymorphisms appear to significantly influence HbF levels and age at presentation in thalassemia major but not in thalassemia intermedia patients. Small numbers precluded a definitive correlation of the polymorphism with response to hydroxyurea therapy.