Thermal Chemosensitization of Breast Cancer Cells to Cyclophosphamide Treatment Using Folate Receptor Targeted Gold Nanoparticles

This article explores the application of hyperthermia mediated by alpha human folate receptor (αHFR) targeted gold nanoparticles (GNPs) for potentiating the cytotoxicity of cyclophosphamide (CPA) in αHFR positive breast cancer cells. Folate functionalized GNPs were delivered to highly αHFR positive breast cancer cells MDA-MB-231 and to MCF-7 breast cancer cells that does not express detectable levels of αHFR followed by hyperthermia. We have shown that hyperthermia induced by folate functionalized GNPs sensitized MDA-MB-231 cells by ten-fold to CPA treatment, whereas MCF-7 cells exhibited only onefold chemosensitization. Collectively, the study suggests the feasibility of using αHFR targeted GNPs for facilitating increased cellula r uptake of CPA in cancer cells expressing elevated αHFR, allowing reduction in drug dosage.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.     

Association of interleukin-18 genotypes (−607C > A) and (−137 G > C) with the hepatitis B virus disease progression to hepatocellular carcinoma

Chronic infection with HBV has been reported to be associated with the development of HCC. The inflammation mounted by cytokine-mediated immune system plays an important role in the pathogenesis of HBV-associated HCC. IL-18 is a pro-inflammatory cytokine whose role in the development of HBV-associated chronic to malignant disease state has not been much studied. The present study was conceived to determine the role of genetic polymorphisms in IL-18, serum levels of IL-18, and expression level of its signal transducers in the HBV disease progression. A total of 403 subjects were enrolled for this study including 102 healthy subjects and 301 patients with HBV infection in different diseased categories. Polymorphism was determined using PCR–RFLP. Genotypic distributions between the groups were compared using odd’s ratio and 95% CI were calculated to express the relative risk. Circulating IL-18 levels were determined by ELISA. Expression levels of pSTAT-1 and pNF?B was determined by western blotting. In case of IL-18(??607C?>?A), the heterozygous genotype (CA) was found to be a protective factor while in case of IL-18(??137G?>?C) the heterozygous genotype (GC) acted as a risk factor for disease progression from HBV to HCC. Moreover, serum IL-18 levels were significantly increased during HBV disease progression to HCC as compared to controls. Also the levels of activated signal transducers (pSTAT-1 and pNF-κB) of IL-18 in stimulated PBMCs were significantly increased during HBV to HCC disease progression. These findings suggest that IL-18 has the potential to act as a biomarker of HBV-related disease progression to HCC.

     

Reactivation of fatty acid oxidation by medium chain fatty acid prevents myocyte hypertrophy in H9c2 cell line

Molecular and Cellular Biochemistry - Metabolic shift is an important contributory factor for progression of hypertension-induced left ventricular hypertrophy into cardiac failure. Under...     

Physiological Slowing and Upregulation of Inhibition in Cortex Are Correlated with Behavioral Deficits in Protein Malnourished Rats

Protein malnutrition during early development has been correlated with cognitive and learning disabilities in children, but the neuronal deficits caused by long-term protein deficiency are not well understood. We exposed rats from gestation up to adulthood to a protein-deficient (PD) diet, to emulate chronic protein malnutrition in humans. The offspring exhibited significantly impaired performance on the ‘Gap-crossing’ (GC) task after reaching maturity, a behavior that has been shown to depend on normal functioning of the somatosensory cortex. The physiological state of the somatosensory cortex was examined to determine neuronal correlates of the deficits in behavior. Extracellular multi-unit recording from layer 4 (L4) neurons that receive direct thalamocortical inputs and layers 2/3 (L2/3) neurons that are dominated by intracortical connections in the whisker-barrel cortex of PD rats exhibited significantly low spontaneous activity and depressed responses to whisker stimulation. L4 neurons were more severely affected than L2/3 neurons. The response onset was significantly delayed in L4 cells. The peak response latency of L4 and L2/3 neurons was delayed significantly. In L2/3 and L4 of the barrel cortex there was a substantial increase in GAD65 (112% over controls) and much smaller increase in NMDAR1 (12-20%), suggesting enhanced inhibition in the PD cortex. These results show that chronic protein deficiency negatively affects both thalamo-cortical and cortico-cortical transmission during somatosensory information processing. The findings support the interpretation that sustained protein deficiency interferes with features of cortical sensory processing that are likely to underlie the cognitive impairments reported in humans who have suffered from prolonged protein deficiency.     

Incidence of Subclinical Mastitis and Prevalence of Major Mastitis Pathogens in Organized Farms and Unorganized Sectors

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10⁵/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.     

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S–5.8S–26S and 5S ribosomal RNA gene families,and the two tandemly repeated DNA sequences

In the genus Carthamus (2n = 20, 22, 24, 44, 64; x = 10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S–26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S–26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n = 20, and the two botanical varieties of B genome C. tinctorius (2n = 24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S–26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n = 20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n = 44), and C. lanatus is one of the progenitors for the hexaploid (2n = 64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2–10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.     

Conformational flexibility in the allosteric regulation of human UDP-α-D-glucose 6-dehydrogenase

UDP-α-D-xylose (UDX) acts as a feedback inhibitor of human UDP-α-D-glucose 6-dehydrogenase (hUGDH) by activating an unusual allosteric switch, the Thr131 loop. UDX binding induces the Thr131 loop to translate ~5 ? through the protein core, changing packing interactions and rotating a helix (α6(136-144)) to favor the formation of an inactive hexameric complex. But how does to conformational change occur given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The 2.3 ? resolution crystal structure of the deletion construct (Δ132) reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. Sedimentation velocity studies show that the open conformation stabilizes the Δ132 construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDX-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. We show that the Δ132 and UDX-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. On the basis of (i) main chain flexibility and (ii) a model of the conformational change, we propose that hinge bending can occur as a concerted motion between adjacent subunits in the high-symmetry hexamer. We combine these results in a structurally detailed model for allosteric feedback inhibition and substrate--product exchange during the catalytic cycle.     

Diet-induced impaired glucose tolerance and gestational diabetes in the dog

Glucose metabolism was compared in dogs consuming a chow/meat diet throughout pregnancy (P group, n = 6) and dogs switched to a high-fat/high-fructose (HFF) diet during the 4th-5th gestational week (gestation ?9 wk; P-HFF group; n = 6). An oral glucose tolerance test (OGTT; 0.9 g/kg) was administered in the 6th-7th gestational week, and a hyperinsulinemic [0-120 min: 1.8 pmol·kg(-1)·min(-1) (low insulin); 120-240 min: 9 pmol·kg(-1)·min(-1) (high insulin)] euglycemic clamp was performed the following week. Nonpregnant (NP) female dogs underwent OGTTs but not clamp studies. All P-HFF dogs exhibited impaired glucose tolerance (IGT) or gestational diabetes (GDM), but only one P dog had IGT. Insulin concentrations in P and P-HFF dogs were significantly lower than in NP dogs 30 and 60 min after the OGTT. Therefore, mean islet size and area were evaluated in P and NP dogs. These values did not differ between groups, and proliferating endocrine cells were rare in pregnancy. During exposure to high insulin, glucose infusion rate and hindlimb glucose uptake were ～30% greater (P < 0.05) and net hepatic glucose output was more suppressed (-5.5 ± 6.1 vs. 7.8 ± 2.8 mg·100 g liver(-1)·min(-1), P < 0.05) in P than in P-HFF dogs. In conclusion, in the 2nd trimester the canine pancreas does not exhibit islet hypertrophy, hyperplasia, or neogenesis. Combined with the lack of pancreatic adaptation, a HFF diet during late pregnancy produces a canine model of IGT and GDM without hyperinsulinemia but exhibiting liver and muscle insulin resistance.