bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of Gut Micro‐flora of Mice Fed Dietary Resistant Starch
Authors: Bae Hee‐Sung , Hou Aixin , Martin Roy J , Zhou Jun , Keenan Michael J
Published: June 2021

Microorganisms in the human gut reach 10 to 100 trillion, and their metabolic activities impact health. Resistant starch (RS) is a fermentable fiber which promotes fermentation in the lower gut. This study investigated how RS impacts the gut microbial community structure. We characterized the gut microflora in mice fed dietary RS using molecular approaches. Total bacterial DNA was isolated from the cecal contents of 2 groups of mice (n=3) fed RS or an equal energy density diet. 16S rRNA was amplified by PCR, cloned, and randomly selected clones were sequenced. Most sequences (≥89%) observed from these treatments were affiliated with two bacterial groups, Firmicutes and Bacteroidetes. Phylum Firmicutes revealed a higher diversity (≥180 operational taxonomic units, OTU), than phylum Bacteroidetes (≥30 OTUs). Firmicutes groups also showed obvious differences in the composition of subgroups based on their phylogenetic relationships between RS and control, while the Bacteroidetes presented a similar composition between the diets. Larger portions (≥50%) of two species belonging to family Lachnospiraceae in the phylum Firmicutes were exclusively detected with RS, implying that these organisms may be closely involved in the RS metabolism. The results provide molecular information on shifts in gut microbial community structures impacted by RS. Supported by LSU AgCenter.
mir30e is a downstream target for the beta‐catenin/TCF4 transactivation pathway
Authors: Liao Yalin , Lönnerdal Bo
Published: June 2021

Maintenance of the small intestinal epithelium is based on a supply of specialized villous cell lineages differentiated from progenitors in the crypts. The Wnt/beta‐catenin/TCF4 signaling pathway plays critical roles in this process, but little is known about its downstream targets. MicroRNAs (miRNAs) are endogenous, non‐coding RNAs ~22 nt in size, down‐regulating protein translation by binding to their target mRNA 3'UTRs. We identified mir‐30e as an immediate downstream target activated by beta‐catenin/TCF4. Bioinformatics analysis revealed clustered beta‐catenin/TCF4 binding sites within a 2000 bp promoter region of mir‐30e gene for human, mice, and rats. This promoter region was cloned into pGL3‐control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV‐SPORT6‐beta‐catenin expression vector dose‐dependently increased luciferase activity, and co‐transfection of pCMV‐SPORT6‐TCF4 further enhanced promoter activity. Dexamethasone induced differentiation of IEC‐6 cells caused a 2‐fold increase in mir‐30e expression, and upon beta‐catenin siRNA transfection, mir‐30e increased 1.3‐fold. Binding of beta‐catenin/TCF4 complexes from IEC‐6 nuclear extracts to the 4 putative sequences in the human mir‐30e promoter was confirmed by EMSA. These results demonstrate that beta‐catenin/TCF4 transactivates mir‐30e during intestinal cell differentiation.

Grant Funding Source

Internal
Modulating the gut flora alters amino acid metabolism in neonatal pigs
Authors: Puiman Patrycja , Burrin Douglas , van Goudoever Johannes , Boehm Günther , Stoll Barbara
Published: June 2021

Objective

Intestinal microbes consume and produce amino acids (AA). This may impact intestinal threonine (THR) metabolism necessary for adequate gut function. We hypothesized that modulating the gut flora results in an alteration of intestinal THR utilization and hence whole body AA metabolism.

Methods

Neonatal pigs were randomized to one of 3 groups (n=7/group): Control (C), Probiotics (PRO) or antibiotics (AB). After 9 days, piglets received an isotope tracer infusion with intravenous (iv) [15N2] Urea, and 2 days later with iv [1‐15N] THR and intragastric [U‐13C, 1‐15N] THR. Piglets were euthanized and blood, intestinal contents and tissue samples collected.

Results

Modulating the gut flora resulted in lower gut weight, protein and DNA concentration, and protein synthesis rate in AB and PRO. PRO increased liver weight, but lowered liver DNA concentration. AB decreased urea flux and increased plasma levels of urea cycle products ornithine and citrulline. In AB, THR flux and plasma levels were increased. THR splanchnic uptake was similar amongst treatment groups.

Conclusions

Modulating the microbial flora decreases gut protein metabolism and affects the urea cycle. Alterations in total AA metabolism may be due to decreased protein breakdown or decreased urea production from ammonia. This might impact growth and health in neonates that have a high protein requirement and are often treated with antibiotics.
Crystal structure of SGLT reveals mechanism of Na+/sugar cotransport
Authors: Abramson Jeff
Published: June 2021

Membrane proteins that convert energy stored in electrochemical sodium gradients to drive nutrients into cells constitute a major class of proteins widely distributed throughout all kingdoms of life. We recently reported the crystal structure of a representative member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). This transporter is homologous to human members of the SSS family that are important in health and disease. The ~3.0′ structure shows 14 transmembrane helices with a core structure composed of inverted repeats of 5 TM helices (TM2‐TM6 and TM7‐TM11). In the center of the core resides a bound galactose that is occluded, by hydrophobic residues, from a large aqueous cavity open to the intracellular milieu, representing the inward‐facing conformation. The architecture of the core is similar to both the leucine transporter (LeuT) and the more recently reported Benzyl‐hydantoin transporter (Mhp1) from different gene families. Modeling the outward‐facing conformation based on LeuT amd Mhp1 structures, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.
Effect of cadmium on heme oxygenase‐1 during cellular iron deficiency
Authors: Lai Chengzhi , Loo George
Published: June 2021

Trace metals can have contrasting biological effects. Iron and cadmium are normally regarded as a crucial micronutrient and noxious environmental agent, respectively. However, it is unknown if iron is required to help defend cells against cadmium. Thus, the effect of cadmium on cytoprotective heme oxygenase‐1 (HO‐1) during cellular iron deficiency caused by iron chelators (deferoxamine and 2,2′‐dipyridyl) was investigated in HCT‐116 cells. Exposing cells to cadmium induced HO‐1 enzyme activity and increased HO‐1 protein expression, which were attenuated by thiol antioxidants and also by chemical inhibitors of the flavoheme protein, NADPH oxidase (NOX). In iron‐deficient cells exposed to cadmium, induction of HO‐1 mRNA, protein, and enzyme activity were all blunted. Also, nuclear translocation of the redox‐sensitive transcription factor, Nrf2, which is known to activate the HO‐1 gene, was diminished. But, after treating the iron‐deficient cells with ferrous sulfate to restore positive iron balance, all of the above molecular events were not impaired upon exposing the rescued cells to cadmium. Zinc or copper treatment was ineffective. These results suggest that iron, being a critical element of the NOX catalytic subunit, plays a pivotal indirect role in the capacity of cadmium and potentially other xenobiotics to optimally upregulate HO‐1 gene expression. Supported by NRI USDA CSREES (2006‐35200‐16578).
Effects of iron and/or zinc deficiency on blood biochemical parameters in rats
Authors: Konomi Aki , Yokoi Katsuhiko
Published: June 2021

To investigate the effects of iron (Fe) and zinc (Zn) deficiency on blood biochemical parameters (plasma lipids, protein, plasma AST, ALT and ACE activities), forty 4‐week‐old male Sprague‐Dawley rats were assigned into 4 dietary treatment groups of 10 for the 4‐week study: severely Fe‐deficient group (FD), moderately Zn‐deficient group (ZD), severely Fe‐deficient/moderately Zn‐deficient group (FZD), and control group (Control). After 4 weeks on dietary regimen, rats were fasted overnight and anesthetized with diethylether. Blood was collected from the abdominal aorta with heparin. Statistical analysis was conducted using Tukey's simultaneous multiple comparison test. Plasma TG, T‐Cho and FFA concentrations were not significantly different among all treatment groups. Plasma PL concentration of FD and FZD was significantly higher than that of ZD. Compared with Control, plasma total protein and globulin concentrations of FD were significantly higher, and those of ZD tended to decrease. However, the Fe deficiency‐associated alterations were suppressed in the combined deficiency of Fe and Zn. Plasma albumin concentration was not significantly different among all treatment groups. The A/G ratio of FZD was significantly higher than FD. Plasma AST activity of FD was significantly higher than that of Control. Plasma ACE activity of FD, ZD and FZD were significantly higher than that of Control. These data indicate an interaction between Fe and Zn metabolism.
Using the domestic chicken (Gallus gallus) as an in vivo screening tool for Fe bioavailability
Authors: Tako Elad , Rutzke Mike A , Glahn Raymond P
Published: June 2021

Fe fortification of foods and biofortification of staple food crops are strategies that alleviate Fe deficiency. The broiler chicken may be a useful model for initial in vivo screening of Fe bioavailability in foods due to its growth rate, anatomy, size and low cost. In this study, we assess the broiler as a model for hemoglobin repletion studies (HRE) and present a unique duodenal loop technique for direct measurement of intestinal Fe absorption. One week old chicks were divided into Fe deficient vs. Fe adequate treatment groups. For 6 weeks, hemoglobin (Hb), feed consumption and body weights (BW) were measured. At week 7, birds were anesthetized and their duodenal loops were exposed. The two halves of the loop were isolated and a catheter was inserted into the duodenal vein. A solution containing either 54Fe or 58Fe (1mg in10mM ascorbic acid) was injected into half of the loop. Blood samples were collected every 5 min and for 90 min post injection and analyzed by ICP‐MS for 58Fe and 54Fe concentrations. In the Fe‐deficient group, Hb concentrations, Hb‐Fe and BW were lower and HRE was higher than in the Fe‐adequate group (P<0.05). Within groups, no differences in Fe absorption were shown between the two halves of the loop; however, Fe absorption was higher in the Fe‐deficient birds (P<0.05). These results indicate that this model can serve as an intermediate screening tool for Fe availability (i.e. between in vitro and human studies)
Zinc status in gastroenterological surgical patients. Association with complications
Authors: Zago Liliana , Danguise Eduardo , González Infantino Carlos , Callegari Mariana , Río María Esther
Published: June 2021

The relevant importance of Zn, an essential micronutrient, in the surgical patient is well recognized. There is little biochemical information about deficiency in Argentina and dietary data of ENNyS (national survey) showed 33.5% of low intakes. In this study plasma levels, basal and 24 hs excretion of Zn were evaluated in 46 patients previous to programmed gastroenterological surgeries. Plasma Zn was 84.5±26.2 μg/dl; distribution shows 22% of deficient (<70), 24% marginal (70‐85) and 54% of adequate values (>85 μg/dl). Urinary excretion was 588±463 μg/day; 31% <300, but only 8% <200 μg/day. Basal excretion was 0.61±0.51 mg Zn/g creatinine; 38% <0.4 mg/g, compatible with low intakes. About plasma Zn, women presented lower values than men (81.2±27.2 vs 87.6±25.5 μg/dl); an increase with BMI was observed: 59.8±3.2 in undernourished, 82.4±21.9 in normal, 84.9±31.1 in overweight and 99.0±35.4 μg/dl in obese, in opposition with decreased values described for obese by other authors; patients with inflammation (PCR>0.5 mg/dl) presented decreased values (79.6±32.1 vs 89.8±16.), and in those with PCR>4 fall to 55.0±25.9 µg/dl. Postoperative complications appeared in 67% of patients with values <70, and in 32% of those >70 µg/dl (Relative Risk= 2.06; Odds Ratio=4.18). All studied markers agree that Zn status must be evaluated previous to surgery because of the magnitude of deficiency and the higher rate of complications that deficiency exposes the patient. Supported by UBACyT B090.
Attenuation of ZnT2 in tumorigenic breast cancer cells results in apoptosis
Authors: Foolad Farnaz , Lopez Veronica , Kelleher Shannon L
Published: June 2021

Zinc (Zn) levels are elevated in breast tumor biopsies. Regulation of mitochondrial Zn metabolism controls intrinsic apoptosis, which is dysregulated in breast cancer. ZnT2 imports Zn into vesicles and mitochondria in breast cells. Our objective was to determine if altered ZnT2 expression in breast cancer cells affect the apoptotic pathways. To explore functional differences, we first determined that tumorigenic breast cancer cells (T47D) have higher cellular Zn levels compared with normal breast cells (HC11). Interestingly, ZnT2 protein abundance was 10‐fold lower in tumor cells. Mitochondrial Zn levels (RhodZin‐3 fluorescence) were 30% lower in tumor cells. ZnT2‐attenuation in cancer cells reduced mitochondrial Zn pools further by ~30%. This resulted in a ~40% decrease in cell viability in tumor cells which did not occur in normal cells. Decreased cell number was associated with increased Annexin‐V staining, illustrating that tumor cell death resulted from apoptosis and not necrosis. In conclusion, our results suggest that under‐expression of ZnT2 in tumor cells reduces the ability of breast cells to modulate mitochondrial Zn level to regulate the apoptotic threshold. Statistics indicate that >200,000 new cases and deaths result from breast cancer annually, thus development of new and innovative therapies is critical. Results from our study suggest that ZnT2 may be a potential therapeutic target.
Increased plasma concentrations of soy isoflavones by increased doses of soy isoflavones are irrelevant to lipid peroxidation and aberrant crypt foci formation in colonic cancer model rats
Authors: Choi Young‐Sun , Min Won‐Kyung
Published: June 2021

We have investigated whether soy isoflavones supplemented to diets increase plasma concentrations of soy isoflavones and equol, and whether increased plasma concentration of soy isoflavones suppresses tissue levels of lipid peroxides and aberrant crypt foci (ACF) formation in colon cancer model rats. Sprague Dawley male rats were injected with 1,2‐dimethylthydrazine (DMH) and given one of AIN93‐based experimental diets (0, 10, 50, 150, or 500 mg isoflavones per kg diet) for 12 weeks. Thiobarbituric acid reactive substances (TBARS) of plasma and homogenates of liver and kidneys were determined using TBARS assay kit. Plasma concentrations of genistein, daidzein and equol were determined using kits by competitive time‐resolved fluoroimmunoassay technique. Plasma concentrations of genistein (R2=0.521 by genistein intake, p<0.001), daidzein (R2=0.623 by daidzein intake, p<0.001) and equol (R2=0.528 by daidzein intake, p<0.001) were increased dose‐dependently. However, the numbers of ACF decreased significantly (p<0.01) in 10 or 50 mg isoflavones‐fed groups as compared with the control group, and there was no significant correlation between the number of ACF and plasma concentration of genistein, daidzein or equol. Plasma TBARS level was not influenced by either dose or plasma level of soy isoflavones. Hepatic levels of TBARS were significantly lower in 10 and 50 mg isoflavones‐fed groups as compared with the control group. The level of kidney TBARS was significantly lower in only 50 mg isoflavones‐fed group as compared with the control group. Therefore, the results indicate that optimal level of soy isoflavones seemed to be most effective in suppressing ACF formation and lipid peroxidation in DMH‐induced colon cancer model rats.
Neuroprotective effect of isoflavones against homocysteine‐mediated neuronal degeneration in SH‐SY5Y cells
Authors: Kwon Young Hye , Park Youn‐Jin , Jang Yu‐mi
Published: June 2021

Several studies reported that ER stress‐induced homocysteine (Hcy) toxicity may play an important role in the pathogenesis of neurodegeneration. Estrogen as well as isoflavones has been suggested to have the protective effect against the risk of developing neurodegenerative diseases. Therefore, in the present study, we investigated effects of isoflavones (genistein and daidzein) on Hcy‐mediated neurotoxicity in SH‐SY5Y human neuroblastoma cells. Treatment of cells with isoflavones significantly protected cells against Hcy‐mediated apoptosis. We observed significant increase in DNA damage by Hcy, which was alleviated by isoflavones. Isoflavones suppressed Hcy‐mediated ER stress as determined by decreased expressions of the binding protein (BiP) mRNA, spliced X‐box‐protein (Xbp)‐1 mRNAs and C/EBP homologous transcription factor (CHOP) protein. Treatment of cells with isoflavones also significantly inactivated GSK3β and decreased tau hyperphosphorylation. In conclusion, our results demonstrate that isoflavones may alleviate ER stress‐mediated neurodegeneration. This study was supported by a grant from Korea Research Foundation.
Epigenetic expression of annexin A1 and cytostatic effects of HDAC inhibitors in breast adenocarcinoma cell line, MCF 7
Authors: Hirata Aiko , Senanayake Thulani , How Yingchun , Woster Patrick W , Hirata Fusao
Published: June 2021

Annexin A1 is a member of the protein family that binds to phospholoipids in a Ca2+ dependent manner, and from its chemical nature is thought to be closely associated with membrane functions such as membrane organization, trafficking and metabolism. On the other hand, this protein is a major substrate of oncogenic kinases such as c‐met and c‐src, and is thus proposed to be involved in signal transduction of growth factors and mitogens. Since the expression of annexin A1 is decreased in breast cancer and prostate cancer, this protein is thought to have supressor functions in these cancers. In accord with this interpretation, its expression was increased, when breast adenocarcinoma MCF 7 cells were treated with THS‐79‐12, a polyaminobenzamide histone N‐deacetylase (HDAC) Class 1 inhibitor. The epigenetic induction of annexin A1 paralleled the inhibition of HDAC. The cell growth of THS‐79‐12 treated MCF 7 cells was arrested, and their morphology became similar to that of normal breast epithelial cells, MCF 10A. The cell morphology and cell growth of MCF 7 cells transfected by cDNA encoding human annexin A1 exhibited similar changes with those of THS‐79‐12 treated cells. Roles of annexin A1 in apoptosis pathway and EGF signal transduction will be discussed. (Supported in parts by a grant from The Susan G. Komen Breast Cancer Foundation.)
Immunohistochemical expression of pituitary tumor transforming gene in pituitary adenomas: clinico‐pathological variables
Authors: Salehi Fateme , Manoranjan Branavan , Scheithauer Bernd W , Lloyd Ricardo V , Kalman Kovacs , Cusimano Michael D
Published: June 2021

Pituitary tumor transforming gene (PTTG) was initially found to be overexpressed in GH4 rat pituitary adenomas (PA). PTTG overexpression has been correlated with patient survival, tumor grade, and/or invasiveness in several tumors including those of thyroid, breast, lung, and colon. Studies of PTTG as a prognostic indicator in PAs are limited and show conflicting results. We investigated PTTG immunostaining in 79 PAs of different subtypes (growth hormone (GH), 12; prolactin (PRL), 6, adrenocorticotropic hormone (ACTH), 11, gonadotroph, 21; null cell, 29) in relation to clinico‐pathological variables including patient age and sex, as well as tumor size, and invasiveness. The streptavidin‐biotin‐peroxidase method was used, with a monoclonal PTTG antibody (Abcam, Cambridge, UK). PTTG expression was found to be cytoplasmic in all PAs, with highest levels being present in GH adenomas and lowest in the PRL ones. No correlation was found with patient age, sex, tumor size or invasiveness. Thus, it appears that PTTG cannot serve as a prognostic marker in patients with PAs. PTTG has been implicated in several tumorigenic processes including angiogenesis, DNA damage repair and invasiveness. PTTG overexpression in PA warrants further research into its role in PA initiation and progression.
+ACA BRCA1 promoter polymorphism genotypic frequency among unaffected individuals and breast disease patients
Authors: White Kristen K. , Basta Patricia V. , Booker Jessica K , Highsmith W. Edward , Tsongalis Gregory J. , Millikan Robert C. , Coleman William B
Published: June 2021

We previously identified a +ACA polymorphism at ‐600 bp in the BRCA1 promoter that creates a FAC1 transcriptional repressor binding site. Subsequently, we performed a genotyping study to determine the prevalence of this BRCA1 promoter polymorphism among unaffected individuals and patients with breast disease. The genotypic distribution of the BRCA1 promoter was determined for 997 unaffected individuals and 763 patients with breast disease from the Mayo Clinic, UNC Hospitals, and the Carolina Breast Cancer Study. The BRCA1 promoter genotypic frequency among unaffected individuals was 481/997 (48%) WT/WT, 432/997 (43%) WT/ACA, and 91/997 (9%) ACA/ACA. The BRCA1 promoter genotypic frequency among patients with breast disease was 348/763 (46%) WT/WT, 343/763 (45%) WT/ACA, and 72/763 (9%) ACA/ACA. No differences were noted in the genotypic distribution of the BRCA1 promoter among patients with DCIS, BRCA1 mutation, suspected BRCA1‐related cancer (BRCAx), or sporadic breast cancer. The allelic frequency of the +ACA BRCA1 promoter polymorphism among breast disease groups [343/415 (83%) WT/ACA and 72/415 (17%) ACA/ACA] were not different than the allelic frequency of the controls [432/523 (83%) WT/ACA and 91/523 (17%) ACA/ACA]. Our results suggest that the +ACA BRCA1 promoter polymorphism occurs commonly, and may contribute to breast cancer upon activation of the FAC1 repressor protein. Support: NIH CA78343.
A test of HPLC cation exchange resolution of venom major peaks
Authors: Price Joseph A. , Sanny Charles G.
Published: June 2021

To find a first step method for high resolution multiple dimension compositional analysis of venom proteomes using mild conditions to preserve enzymatic activity, a systematic analysis of HPLC cation exchange resolution of venom major peaks was done in the pH range 7.5‐5.5, which has not been previously reported. Test Crotalid venoms (A. p. piscivorus, C. adamanteus, C. atrox) loaded on the column with the void peak ranging from 35‐75% of total chromatogram absorbance. The amount of column loading varied substantially with pH for C. atrox, but not for the others. The loaded material resolved during elution gradient to varying extents, typically into 4‐6 peaks of 5% or greater of total absorbance and many small peaks. This approach's data, new to proteomics of snake venom, suggest that when optimized with column choice and elution salt, this methodology using mild acidic conditions to preserve activity will prove to be a useful first step in a 2‐D chromatographic separation of whole venoms, a key step in elucidating the biochemistry of unknown venom agents.

This work was done with departmental funds.
Histologic characterization of a breast adenocarcinoma experimental metastasis model in SCID beige mice for anti‐neoplastic immunotherapeutic studies
Authors: Egenolf Devon D , Rafferty Patricia , Wiley Diana , Millar Hillary J , Brugger Michelle , Ferrante Catherine , Shi Frank , McCabe Francis , Snyder Linda , Bugelski Peter , Johns Laura
Published: June 2021

Tumor histopathology is an important method to understand the mechanism of action of anti‐neoplastic therapies and immunotherapeutics in animal models. An experimental metastasis model was established with the MDA‐MB‐231 human breast adenocarcinoma cell line in SCID beige mice. Compared to subcutaneous and orthotopic models, this model allows evaluation of multiple lesions at earlier time points. Mice were injected i.v. with MDA‐MB‐231 cells and sacrificed either 14 or 21 days post tumor cell injection. Lungs were processed for histology and stained with H&E. Multiple focal lesions were observed and these lesions had a random distribution in the lung and were variable in size. The lesions increased in size and number over time. These observations were confirmed by a rank order analysis on disease severity, and morphometry on size of the lesions. Morphometry also revealed a significant increase in proliferating cells within lung lesions at Day 21 compared to Day 14. Numerous infiltrating macrophages could also be identified within the lesions. No evidence of angiogenesis within the lesions was observed in this time course. It is hypothesized that the lesions studied have not yet crossed the angiogenic switch and therefore no new blood vessel formation was observed. Lesions can be further characterized by additional histologic methods.
New orthotopic mouse models and Ad‐based transcriptionally targeted vectors for squamous cell carcinoma therapy development
Authors: Zolochevska Olga , Figueiredo Marxa L
Published: June 2021

Oral squamous cell carcinoma affects about 30,000 Americans each year, and although the overall prognosis has improved in recent years for localized carcinomas, metastases can decrease survival rates by half. For tongue and palate lesions, surgery is usually the initial treatment, but distant metastases can develop at sites where conventional therapies are ineffective (lung, bone, heart). Therefore, our goal has been to develop a fast‐growing orthotopic stromal‐epithelial SCC model with a metastasis profile, in which to test new adenoviral‐based gene therapies. We specifically plan to use systemic delivery of an adenovirus (Ad) that targets expression of therapeutic gene p12/CDK2AP1 to SCC cells using the SCCA2 promoter and a transcriptional amplification system. We developed new orthotopic stromal‐epithelial SCC models for floor of mouth and tongue carcinoma with rapid latency and partial tumor spread to regional lymphnodes. We constructed and are characterizing an Ad‐TSTA‐p12FL bidirectional vector for gene therapy applications. We plan to test in vivo the specificity of the vector, the effectiveness in tumor growth inhibition at both floor of mouth, orthotopic, or distant sites, and following intratumoral or systemic administration. We propose this vector may hold promise as a specific, novel, and robust SCC therapy to treat tumors that have metastasized to distant and thus difficult to treat sites.
The SNP276G>T polymorphism in the adiponectin gene is associated with the metabolic syndrome in patients with type 2 diabetes mellitus in Korea
Authors: Hwang Ji‐yun , Park Hee Jung , Choi Young Ju , Huh Kap Bum , Kim Wha‐Young
Published: June 2021

The SNP276G>T polymorphism in the adiponectin gene was reported to be associated with the metabolic syndrome in Koreans. However, few studies have investigated such an association in patients with type 2 diabetes mellitus (DM). Subjects were 754 type 2 DM patients recruited from a DM clinic in Seoul, Korea. The patients with known CVD were excluded. The adiponectin SNP276 genotypes were analyzed. The genotype frequencies of SNP276 (GG/GT/TT) were 45.07%, 46.86%, and 8.07%. Multiple logistic regression analysis revealed that the OR for the metabolic syndrome was greater in subjects with the TT genotype than with GG genotype (OR=2.130; 95% CI=1.128‐4.025) after adjustment for age, sex, obesity (BMI=25), and carbohydrate intake as a percentage of the total energy intake. This trend did not change after further adjustment of serum adiponectin levels (OR = 1.983; 95% CI = 1.038‐3.788). The association between serum adiponectin levels and the risk of metabolic syndrome was examined within each genotype. Only in the GT genotype, serum adiponectin was negatively associated with the risk of metabolic syndrome (OR=0.329; 95%CI=0.227‐0.477). The SNP276G>T polymorphisms may play an important role in the pathogenesis of metabolic syndrome in Korean patients with type 2 DM. (Supported by the Second stage of BK21 Project in 2008 and Seoul R&BD Program, No. 10526)
Expression of TLR9 in Horse, Cow, and Human Lung
Authors: Schneberger David , Caldwell Sarah , Suri Sarabjeet Singh , Kanthan Rani , Singh Baljit
Published: June 2021

Toll‐like receptor 9 (TLR9) is the receptor used for responding to bacterial DNA in many species. Recent work has shown that this receptor is expressed in a diverse set of cells within the lung. However, much of this work has been done in tumors, explanted cells, or cell cultures. We examined the in situ expression of TLR9 in the lung of cattle, horses and humans. The data show expression of TLR9 in monocytes, airway epithelial cells, and capillary endothelium in the lungs of all the species. Pulmonary intravascular macrophages of the horse and cattle also express TLR9. Immno‐electron microscopy revealed TLR9 expression in the cytoplasm and, more interestingly, in the nucleus of pulmonary intravascular macrophages (PIMs) and other lung cells. Lung inflammation as a result of E. coli LPS (horses), Mannheimia hemolytica infection (calves), or sepsis (human) resulted in an influx of TLR9‐positive cells. Finally, we show that the LPS‐induced increase in TLR9 gene transcription in the horse lung was abrogated by pre‐treatment with gadolinium chloride to deplete PIMs which demonstrates PIMs as a major source of TLR9 in the horse lung. We conclude that TLR9 is constitutively expressed in lungs of horse, cow and human. The implications of TLR9 expression in PIMs in the horse and cow for infections need to be clarified. (NSERC)

Grant Funding Source

NSERC
Regulation of obesity, heart function, and lifespan by the nutrient sensing TOR pathway
Authors: Oldham Sean Michael , Birse Ryan , Diop Soda , Graham Suzanne , Bodmer Rolf
Published: June 2021

Metabolic homeostasis is a highly regulated process, with dysregulation causing increased susceptibility to diseases like obesity, diabetes, cardiovascular disease, and cancer. Unfortunately, solving this biomedical problem is difficult because of the polygenic and multi‐organ nature of metabolic homeostasis. Here we establish Drosophila as a genetic model to study the genes and environmental interactions responsible for mediating High Fat Diet (HFD) induced obesity. We see that the nutrient sensing pathway Target of Rapamycin (TOR) is activated and that loss of TOR function can block the detrimental effects of a HFD. We then examined heart function to see if HFD‐induced heart defects can be autonomously and non‐autonomously blocked by TOR. We found that alteration of TOR function in the adipose tissue produced beneficial effects on heart function. This would suggest that TOR regulated peripheral effects like alterations in metabolites, hormone and lipid regulation may be key factors in regulating the adverse effects of HFD. We also examined the effects of altering TOR function both systemically and tissue specifically on feeding and behavior. Lastly, we show that TOR has additional roles regulating metabolism and lifespan by mimicking Dietary Restriction (DR). These phenotypes include lowered glucose and lipid levels, increased longevity, and a block in the age‐dependent decline in heart function. Thus, our findings indicate that the TOR pathway is an important mediator of HFD‐induced obesity and aging, and that alterations of TOR activity may be a critical link from HFD‐induced obesity to diabetes and cardiovascular disease.
AMPK: A Critical Metabolic Signal
Authors: Goodyear Laurie J. , Koh Ho‐Jin , Fujii Nobuharu , Toyoda Taro , Witczak Carol A. , Middelbeek Jan Willem , Jung Michelle , Hirshman Michael
Published: June 2021

The AMP‐activating protein kinase (AMPK) and its upstream kinase LKB1 are serine/threonine kinases that have been proposed to function as master regulators for a diverse array of cellular processes. In skeletal muscle, there is emerging evidence that AMPK and LKB1 regulate multiple metabolic functions, including glucose transport. Our group and others have generated mice lacking AMPK activity in skeletal muscle, as well as muscle‐specific LKB1 knockout mice. In this talk, the putative role of LKB1 and AMPK in regulating skeletal muscle metabolism will be discussed. In addition, recent data will be shown on sucrose non‐fermenting AMPK‐related kinase (SNARK), an AMPK‐related kinase that has previously been shown to be regulated by multiple cellular stresses in kidney and several tumor cell lines. This presentation will discuss our work showing that exercise and contractile activity increase SNARK activity in skeletal muscle. Data will also be presented suggesting a role for SNARK in glucose transport regulation in skeletal muscle.

This research is supported by the American Physiological Society Postdoctoral Fellowship in Physiological Genomics (HJK) and NIH grants AR45670 and DK68626 (LJG).
N‐DOMAIN ACE ACTIVITIES AND EXPRESSION PROFILES IN CHRONIC RENAL FAILURE PATIENTS
Authors: ARITA DANIELLE YURI , SAKATA MAISA MAYUMI , WATANABE INGRID KAZUE , PEREZ JULIANA DINEIA , COLUCCI JULIANA ALMADA , MORAIS LUCIENE , CASARINI DULCE ELENA
Published: June 2021

Diabetes mellitus (DM) is recognized as a leading cause of chronic kidney disease and end‐stage renal failure. The local increase of components of Renin‐Angiotensin System has been implied in different hypertension pathogenesis. In our laboratory, we observed N‐domain ACEs isoforms with 190 and 65 kDa in urine of healthy subjects, and 90 and 65 kDa isoforms were detected in urine of hypertensive patients, suggesting that the 90 kDa ACE could be a possible genetic marker of hypertension. The aim is to associate dysfunctions of the renal system with possible alterations in protein expression and/or ACE isoforms enzymatic activity and in angiotensin (ang) levels in chronic renal failure (CRF) patients. Patients were divided into three groups: (I) DM ‐ normotensive patients with type 1 DM and normoalbuminuria (n=30); (II) DMH ‐ hypertensive patients with type 1 DM and microalbuminuria (n=18); (III) DMCR ‐ patients with DM type 1 and CRF (n=11). ACE activity was slightly decreased in serum of DMH and DMCR (DM=124.1±6.4; DMH=98.4±9.8; DMCR=95.2±7.8 nmol/min/mL). ACE isoforms were significantly over‐expressed in urine of DMH group compared with other two groups by Western blotting. High AngII levels were detected in patient serum of DMCR (DMCR=3.32±0.54; DM=0.41±0.04; DMH= 0.58±0.43 nmol/mL) group, therefore, AngI and Ang1‐7 did not present significant alterations. Our results suggest that somatic and N‐domain ACEs expressions could be associated with renal damage. High levels of AngII in DMCR patients suggest this peptide is resulted by alternative pathways. Supported by FAPESP.
Effects of chronic chromium picolinate treatment in obese Zucker rats
Authors: Mozaffari Mahmood , Wimborne Hereward , Liu Jun Yao , El‐Marakby Ahmed
Published: June 2021

Several studies suggest that Cr(Pic)3 confers anti‐inflammatory effects while others have reported a clastogenic effect. Utilizing the severely insulin resistant obese Zucker rats, we tested the hypothesis that chronic Cr(Pic)3 treatment improves glycemic control in association with reductions in markers of oxidative DNA damage (i.e., urinary 8‐hydroxy deoxyguanosine (8‐OHdG)) and inflammation (i.e., urinary monocyte chemoattractant protein‐1 (MCP‐1)). Accordingly, obese Zucker rats were treated with a diet containing 10 mg/kg chromium (as Cr(Pic)3) from 6 weeks to 6 months of age; untreated lean and obese Zucker rats served as controls. The treatment did not improve the glycemic status of obese Zucker rats as indexed by blood glucose, plasma insulin or hemoglobin A1c levels. Similarly, urinary MCP‐1 was not affected by excess dietary intake of Cr(Pic)3. Interestingly, urinary 8‐OHdG excretion was reduced in the treated than untreated obese Zucker rats (p<0.05). However, neither mean arterial pressure nor other indices of renal function (e.g., proteinuria, creatinine clearance or urine osmolality) were affected by chronic treatment with Cr(Pic)3. Taken together, the results suggest that dietary supplementation with Cr(Pic)3 decreases oxidative DNA damage in obese Zucker rats that is not accompanied by changes on blood pressure or glycemic status of the animal. Supported by NIH.
Supratherapeutic Insulin Alters Intracellular pH in Human Kidney Tubular Epithelial Cells
Authors: Kruss Szilvia Ilona , Valyi‐Nagy Klara , Setty Suman
Published: June 2021

Background

The profile of metabolic syndrome includes high fasting sugars and hypertension. Intracellular pH (pHi) is vital for the maintenance of the normal metabolic function of human kidney tubular epithelial cells (HK‐2). In the present study we investigated whether high glucose levels and two common agents that are used to treat metabolic syndrome, insulin and lisinopril have an effect on pHi in cultured HK‐2 cells.

Methods

Immortalized tubular kidney HK‐2 cells were cultured in media containing normal (5 mM) or increased (25 mM) amounts of glucose. Cultures were then exposed to therapeutic or supratherapeutic (3 times higher) doses of insulin and lisinopril and consequent changes in pHi were determined using a SNARF 5‐(and‐6)‐carboxy SNARF®‐1, acetoxymethyl ester agent (Invitrogen, Carlsbad) at 8 days after treatment.

Results

pHi was significantly higher (p<.05) in cells treated with 25 mM versus 5 mM glucose. Supratherapeutic insulin treatment was associated with significantly (p<.01) increased pHi relative to cells cultured in normal and increased glucose. Intracellular pH in the supratherapeutic insulin condition was also significantly elevated (p<.01) relative to lisinopril treated cells.

Conclusions

Increased glucose and supratherapeutic insulin lead to increased (more basic) pHi in human kidney tubular epithelial cells. Altered pHi in these cells may have extensive effects on a variety of cellular functions, including promoting insulin resistance. Furthermore, alkalinic changes could assist the propagation of certain types of cancers which will be the basis of further research in our laboratory.
Omission of extracellular glucose increases antidiabetic drug‐induced vasorelaxations and abolishes sildenafil's attenuation of such relaxations
Authors: Peuler Jacob D. , Phelps Laura E.
Published: June 2021

Sildenafil is used to treat erectile dysfunction in men with type 2 diabetes (T2D). Hypertension is common in T2D but sildenafil attenuates vasorelaxant effects of metformin, pioglitazone and rosiglitazone (The Pharmacologist 46:95, 2004): 3 antidiabetic (AD) drugs often used to improve glucose metabolism in T2D. To assess the glucose dependence of this action, vascular rings from rat tail arteries were pretreated with each AD drug ± sildenafil. After 4 hours, all rings were contracted with norepinephrine (NE). With glucose present, all 3 AD drugs relaxed NE contractions and sildenafil abolished such relaxations. With glucose omitted, relaxant effects of the AD drugs were notably increased and sildenafil failed to abolish them. No such glucose dependence was seen if NE contractions were relaxed by other vasodilators (e.g. nifedipine) known not to influence glucose metabolism. We conclude that, in addition to vascular smooth muscle relaxant mechanisms, these 3 AD drugs also activate contraction‐enhancing mechanisms, which are glucose‐dependent and mask the full potential of the relaxant mechanisms. Thus, sildenafil's attenuation of AD drug‐induced vasorelaxations in the presence of glucose may not only involve interference with their relaxant mechanisms but also facilitation of their potential glucose‐dependent contraction‐enhancing mechanisms.
Potentiation of the depressor effect of acetylcholine by carvedilol
Authors: Simaan Joseph A.
Published: June 2021

Carvedilol, a beta‐1,2 and alpha‐1 adrenoceptor blocker was explored in anesthetized rats in which systolic pressure was measured to determine if it enhances the vasodilator effect of single doses of acetylcholine (0.1, 0.15, 0.2, 0.25, 0.5 ug) by enhancing the availability of nitric oxide. At a maximal cumulative dose(1.364±0.228mg/kg) carvedilol potentiated the depressor effect of acetylcholine by 32‐15% (lowest to highest dose, P<0.01‐0.001). This effect was annulled by prevention of nitric oxide synthesis with nitro‐L‐arginine (15mg/kg) and restoration of the baseline systolic pressure with an infusion of nitroglycerin, a nitric oxide donor. Giving this treatment before carvedilol also prevented its potentiation of acetylcholine effect. Carvedilol may partly enhance the depressor effect of acetylcholine by annulling the reflexly‐mediated sympathetic compensation for the hypotension through its alpha‐1 adrenoceptor blockade. Pretreatment with phenoxybenzamine, a blocker of alpha‐1,2 adrenoceptors and an infusion of angiotensin II to restore basal systolic pressure to baseline had minimal effect on the depressor action of acetylcholine. This was also true with pretreatment with propranolol. It is concluded that carvedilol potentiated the depressor effect of acetylcholine essentially by enhancing the synthesis of nitric oxide. Supported by the American University of Beirut.
Sodium nitrite decreases systemic arterial pressure mediated by xanthine oxidoreductase in the rat
Authors: Golwala Neel , Casey David B , Badejo Adeleke , Dhaliwal Jasdeep S , Murthy Subramanyam N , Kadowitz Philip J
Published: June 2021

Recent studies show that nitrite is reduced to NO and elicits significant vasodilator activity, however, the mechanism responsible for the conversion of nitrite to NO is still uncertain. In the present study, the responses of sodium nitrite upon inhibition of xanthine oxidoreductase (XOR) and aldehyde reductase were investigated in the anesthetized rat. The iv injections of sodium nitrite (0.01 ‐ 0.1 mmol/kg) caused dose related decreases in systemic arterial pressure that were significant in control and in L‐NAME treated rats. The responses were slower in onset than responses to SNP, and the dose response curve of nitrite was 2 log units to the right as that of SNP. The decreases in systemic arterial pressure in response to sodium nitrite were significantly reduced by allopurinol and cyanamide (10 ‐ 25 mg/kg iv) but the inhibitory effects were not additive. Both allopurinol and cyanamide caused small decreases in systemic arterial pressure in response to SNP or the NO donor DEA/NO. The results demonstrate that sodium nitrite has similar efficacy as that of SNP and DEA/NO in decreasing systemic arterial pressure but is less potent by a factor of 30 ‐ 100 and support the hypothesis that the conversion of nitrite to NO in large part is mediated by XOR in the anesthetized rat.
Testosterone relaxes mesenteric microvessels by a non‐genomic mechanism that involves production of peroxynitrite from NOS
Authors: Puttabyatappa Yashoda , White Richard E
Published: June 2021

The increased incidence of cardiovascular disease in men compared with premenopausal women suggests an unfavorable effect of male sex hormone testosterone (T2) on the cardiovascular system. Evidence indicates that T2 exerts acute vasorelaxing effects via non‐genomic mechanisms. These effects involve primarily the vascular smooth muscle (VSM). To date, the mechanism behind the vasodilatory action of testosterone is still under debate. Concentration‐response relationships for T2‐induced relaxation of rat mesenteric microvessels (1pM to 10 ìM) indicate a biphasic effect, and suggest a complex, non‐genomic mechanism of action. Rmax of T2‐induced relaxation is 36.45 ± 6, and was unchaged by actinomycin D, transcription inhibitor. Pretreatment with NOS blockers (L‐NAME, Rmax = 5.31 ± 1.58; L‐NPA, Rmax = 9.28 ± 1.54) did inhibit of T2‐induced microvascular relaxation. Moreover, it appears that T2 stimulates NOS activity via the PI3K‐ Akt pathway. We found that wortmanin, an Akt antagonist, inhibited the effect of T2 and immunoblot experiments confirmed this finding. Lastly, the effect of T2 was inhibited by peroxynitrite scavengers: uric acid (Rmax = 9.94 ± 3.52); FeTPPs (Rmax 15.81 ± 3.14) confirmed by fluorescence measurements. In conclusion, T2 induces relaxation of mesenteric microvessels is associated with production of both NO and superoxide which combine to form peroxynitrite. (supported by HL80402)
Influence of Endogenous Testosterone on Vascular Endothelium‐Leukocyte Interaction in Spontaneously Hypertensive Rats (SHRs)
Authors: Filgueira Fernando Paranaiba , Lobato Núbia Souza , dos Santos Rosângela Aparecida , Oliveira Maria Aparecida , Fortes Zuleica Bruno , Tostes Rita , Carvalho Maria Helena Catelli
Published: June 2021

Aim

Endogenous testosterone exerts influence on the vascular endothelium dysfunction in hypertension, and inflammation may play a important role in the onset and progress of cardiovascular diseases. So, we decided to evaluate the impact of castration on the leukocyte behavior in the SHR mesenteric post‐capillary venules.

Methods and Results

Male SHRs were castrated at 12 weeks of age and the leukocyte behavior was studied in vivo‐in situ by intravital microscopy. It was investigated after 6 weeks of orchidectomy and compared to the ones in the intact group. Castration reduced the number of leukocytes rolling and sticking along the venular endothelium, but not modified migrating after leukotriene B4 stimulation. P‐selectin protein expression (immunohistochemistry) as well as VCAM‐1 and PECAM‐1 gene expression (Real Time PCR) were reduced by castration and might be involved in the reduced rollers and stickers. However no difference was detected in ICAM‐1 protein and gene expression between groups. Venular shear rate, leukocyte and blood flow velocity, venular diameter and leukocyte counts were similar between groups. Castration reduced testosterone levels (EIA Kit) and blood pressure (tail pletismography).

Conclusions

Our results suggest that endogenous testosterone might has proinflammatory effect contributing to the development of vascular alterations in hypertension. FAPESP,CNPq/PRONEX,CAPES
Engineered HGF/SF Variants Promote Angiogenesis
Authors: Sinha Roy Rituparna , Gherardi Ermanno , Sengupta Shiladitya
Published: June 2021

Hepatocyte growth factor/scatter factor (HGF/SF) has been implicated in angiogenesis [Sengupta et al, Arterioscler.Thromb. Vasc. Biol.(2003) 23, 69‐75;Sengupta et al. Circulation.(2003)107,2955‐61]. However, low manufacturing yields as a result of complex multidomain structure and propensity to aggregate, limits its therapeutic use. Based on the structural understanding of a naturally occurring splice variant, NK1, we have rationally engineered a fragment, 1K1 (K132E:R134E) of HGF/SF, which overcomes the above challenges and can be produced in yeast. Crystal structure reveals that it binds to heparin and heparan sulfate glycosaminoglycans and subsequently to MET receptor in a distinct manner from NK1. We demonstrate that 1K1 binds to MET receptor on endothelial cells and induces downstream signaling through MAP and PI3 kinase pathways. Treatment with a MET kinase inhibitor, PHA665752, [10−6 M] blocked the response, while vatalanib [10−6 M], a vascular endothelial growth factor receptor 2 inhibitor, failed to do so. 1K1 induced a concentration‐dependent [10−10 M‐10−8 M] proliferation of endothelial cells and endothelial tubulogenesis when plated on matrigel. Our results indicate that such rational engineering could lead to the development of novel angiogenesis agents, and 1K1 could emerge as a potent agent for therapeutic angiogenesis.