Renal ischemia–reperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct

Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia–reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na+, K+, or Cl−. In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility

Effects of endosperm vitreousness and kernel processing of corn silage on fiber and starch metabolism

Three corn hybrids with different endosperm types (flinty, intermediate, floury) were chopped (nonprocessed: 0.95 cm or processed: 1.91 cm chop at a 1-mm roller clearance) for silage. Kernel processing eliminated whole kernels. Processing reduced the medium particle fraction (0.95 to 1.91 cm) resulting in an increased small particle fraction (\u3c0.95 cm). Floury compared with flinty silage resulted in a greater small particle fraction. Processing numerically increased starch content of small particles, and NDF and ADF content of medium particles. Flinty compared with floury silage increased in vitro and in situ NDF digestibility of small particles. Ruminal digestion kinetics of processed versus nonprocessed, and flinty versus floury silage were compared using a standard and a new macro in situ procedure. The standard procedure showed a reduction in NDF digestibility for floury compared with flinty silage. The macro procedure resulted in an increased DM digestibility for processed compared with nonprocessed silage, and an increased DM digestibility and decreased NDF digestibility for floury compared with flinty silage. Eight cannulated, multiparous (BW = 649 kg) and four primiparous (BW = 638 kg) Holstein cows were used to evaluate effects of kernel processing and endosperm type of corn silage on digestibility and milk production. A replicated 4 x 4 Latin square with a 2 x 2 factorial arrangement of treatments was used. Processing improved daily milk production (9.9%), 4% FCM (8.3%), efficiency of 4% FCM produced (12%), and starch digestibility (8.9%), while reducing (14.7%) NDF digestibility. Floury endosperm corn silage improved efficiency (5.1%) of 4% FCM production, and DM (7.5%), OM (8.3) and starch (6.4%) digestibilities. Milk production and feed efficiency can be improved by kernel processing corn silage. Furthermore, selecting floury verses flinty endosperm hybrids can increase milk production efficiency. Changes in physicochemical characteristics of particles result in improved ruminal and total tract digestibilities with processed and floury endosperm silages. Therefore, when formulating lactating dairy cows diets, endosperm type and processing method need to be considered

Corn and Sorghum Distillers Grains for Finishing Cattle

To evaluate corn and sorghum distillers grains in corn-based finishing diets, 60 crossbred, yearling steers were individually fed one of three finishing diets: dry-rolled corn, corn distillers grain or sorghum distillers grain for 127 days. Distillers grains were fed at 30 percent of the dietary dry matter, replacing dry-rolled corn. Distillers grains increased the final weight, daily gain, feed efficiency, hot carcass weight, fat thickness and yield grade compared with the control. Sorghum distillers grains increased dry matter intake and fat thickness compared with corn distillers grains

Effects of Rumensin Level and Bunk Management Strategy on Finishing Steers

Eight ruminally fistulated, yearling steers (two concurrent 4x4 Latin squares) were used to evaluate dietary Rumensin level (0, 30, 30/40 or 40 g/t), and bunk management strategy (ad libitum or clean bunk: 24- or 14-hour feed access). Rumensin decreased meal size and increased meal frequency without compromising intake. Clean bunk management increased consumption rate, meal size and ruminal pH change and pH variance. Steers with limited feed exposure are at greater risk for subacute acidosis; Rumensin effects consumption favorably for controlling acidosis, especially for cattle with limited feed exposure

Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3' mismatches in forward and reverse primers.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system using primers with mismatches at the 3' ends was developed to determine polymorphisms in IL-10 promoter region. Three previously described biallelic polymorphisms in IL-10 were linked in a 12 reaction PCR-SSP system and the method used to provide genotype data on 233 UK and 166 Polish controls. There are eight possible polymorphic combinations in IL-10 promoter gene but only three were observed in both control groups. Population frequencies of IL-10 genotypes show, in contrast to HLA, that UK and Polish frequencies are remarkably similar

Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies.

The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF alpha), lymphotoxin-alpha (LT alpha) and interleukin-10 (IL-10) genes on stimulated TNF alpha and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn's disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF alpha, LT alpha and IL-10 genes. Production of the TNF alpha and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF alpha gene, three haplotypes of LT alpha and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT alpha-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT alpha-2 haplotypes were associated with higher TNF alpha production in CD patients, and the TNF-4 haplotype was associated with lower TNF alpha production in UC patients. The A allele in the IL-10 promoter region at position -1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF alpha, LT alpha and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls