Marker-assisted introgression of blackmold resistance QTL alleles from wild Lycopersicon cheesmanii to cultivated tomato (L. esculentum) and evaluation of QTL phenotypic effects

Blackmold, caused by the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Previously, we found blackmold resistance in a wild tomato (Lycopersicon cheesmanii) and quantitative trait loci (QTL) for resistance were mapped in an interspecific population. Five QTLs were selected for introgression from L. cheesmanii into cultivated tomato using marker-assisted selection (MAS). Restriction fragment length polymorphism and PCR-based markers flanking, and within, the chromosomal regions containing QTLs were used for MAS during backcross and selfing generations. BC₁ plants heterozygous at the QTLs, and subsequent BC₁S₁ and BC₁S₂ lines possessing different homozygous combinations of alleles at the target QTLs, were identified using DNA markers. Field experiments were conducted in 1998 (with 80 marker-selected BC₁S₂ lines) and 1999 (with 151 marker-selected BC₁S₂ and BC₁S₃ lines) at three California locations. Blackmold resistance was assessed during both years, and horticultural traits were evaluated in 1999. The BC₁S₂ and BC₁S₃ lines containing L. cheesmanii alleles at the QTLs were associated with a large genetic variance for resistance to blackmold and moderate heritability, suggesting that significant genetic gain may be achieved by selection in this genetic material. L. cheesmanii alleles at three of the five introgressed QTLs showed a significant, positive effect on blackmold resistance. A QTL on chromosome 2 had the largest positive effect on blackmold resistance, alone and in combination with other QTLs, and was also associated with earliness, a positive horticultural trait. The other four QTLs were associated primarily with negative horticultural traits. Fine mapping QTLs using near isogenic lines could help determine if such trait associations are due to linkage drag or pleiotropy.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly

An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins.     

Noncovalent chiral domino effect on one-handed helix of nonapeptide containing a midpoint L-residue

We here clarify whether noncovalent chiral domino effect characterized by the terminal interaction of a helical peptide with a chiral small molecule can alter the helical stability of N-deprotected peptides containing an L-residue covalently incorporated into the inner position. Two nonapeptides consisting of the midpoint L-leucine (1) or L-phenylalanine (2) and the achiral helix-forming residues were employed. NMR and IR spectroscopy and energy calculation indicated that both peptides adopt a 3(10)-helical conformation in chloroform. They strongly preferred a right-handed screw sense because of the presence of the midpoint L-residue. These original right-handed screw senses were retained on addition of chiral Boc-amino acid, but their helical stabilities clearly depended on its added chirality. Here, Boc-L-amino acid stabilizes the original right-handed helix, whereas the corresponding Boc-D-amino acid tends to less stabilize or destabilize it. This tendency was not observed for the corresponding N-Boc-protected peptides 1 and 2, strongly suggesting that the N-terminal amino group is required for controlling the stabilization of the original right-handed helix. Therefore, noncovalent chiral domino effect in peptides 1 and 2 can contribute even to the helical stability of a chiral peptide prevailing one-handed helix strongly through the midpoint L-residue. In addition, the N-terminal moiety of a 3(10)-helical peptide was found to generate chiral discrimination in complexation process with racemic additives.     

A membrane protein,TMCO5A,has a close relationship with manchette microtubules in rat spermatids during spermiogenesis

We isolated the transmembrane and coiled‐coil domains 5A (Tmco5A) gene using polymerase chain reaction‐based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum‐nuclear membrane (ER‐NM) of cells as a membrane‐associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER‐NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti‐TMCO5A and the anti‐β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.     

Formation of aberrant TJ strands by overexpression of claudin-15 in MDCK II cells

Claudins are one of the transmembrane proteins found at tight junctions (TJs); they constitute the backbone of TJ strands and comprise a multigene family. Claudins share a YV sequence at the COOH-termini, which is considered to be a ZO-binding motif. Overexpression of claudin-15 (15CL) or claudin-15 tagged with enhanced green fluorescent protein at the NH₂-terminus (EGFP-15CL) induced aberrant strands in MDCK II cells, even though claudin-15 has the ZO-binding motif. Morphometric analysis by freeze-fracture electron microscopy revealed that the mean number of apical TJ strands increased by 47% in EGFP-1CL-expressing cells, 21% in EGFP-15CL-expressing cells, and 28% in 15CL-expressing cells. The number of free-ended apical strands increased remarkably in EGFP-15CL- and 15CL-expressing cells, but not in EGFP-1CL-expressing cells. When MDCK cells expressing EGFP-1CL, EGFP-15CL or 15CL were co-cultured with parent MDCK cells, which express claudin-1 but not claudin-15, EGFP-15CL and 15CL could not be concentrated at the apical junctional region between the heterotypic cells, though EGFP-1CL could. These results suggest that not only binding to ZO-1, but also head-to-head compatibility between claudin species, is involved in organizing claudin proteins at the apical junctional region.     

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

Introduction

Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy.

Materials and methods

Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks.

Results

PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney.

Conclusions

These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.     

Alteration of integrin-dependent adhesion and signaling in EMT-like MDCK cells established through overexpression of calreticulin

Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits α5 and αV, resulting in an increase in the formation of complexes such as α5β1 and αVβ3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.