DnaJ-promoted binding of DnaK to multiple sites on σ32 in the presence of ATP.

The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the E. coli heat shock transcription factor σ(32) as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ(32) complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ(32) complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ(32), even though it has been suggested that DnaK interacts with σ(32) at a molar ratio of 1:1. Two σ(32) mutants, L201D σ(32) and I54A σ(32), which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ(32) complex formation. Pulldown assays demonstrated that the affinity of I54A σ(32) for DnaK was similar to that of wild-type σ(32) in the absence of DnaJ, whereas L201D σ(32) exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ(32) was much higher than that eluted with I54A σ(32). These results indicate that there are multiple DnaK binding sites on σ(32) and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site.

Selective gene expression analysis of muscular and vascular components in hearts using laser microdissection method.

Background. The heart consists of various kinds of cell components. However, it has not been feasible to separately analyze the gene expression of individual components. The laser microdissection (LMD) method, a new technology to collect target cells from the microscopic regions, has been used for malignancies. We sought to establish a method to selectively collect the muscular and vascular regions from the heart sections and to compare the marker gene expressions with this method. Methods and Results. Frozen left ventricle sections were obtained from Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHR-SP) at 24 weeks of age. Using the LMD method, the muscular and vascular regions were selectively collected under microscopic guidance. Real-time RT-PCR analysis showed that brain-type natriuretic peptide (BNP), a marker of cardiac myocytes, was expressed in the muscular samples, but not in the vascular samples, whereas α-smooth muscle actin, a marker of smooth muscle cells, was detected only in the vascular samples. Moreover, SHR-SP had significantly greater BNP upregulation than WKY (P < 0.05) in the muscular samples. Conclusions. The LMD method enabled us to separately collect the muscular and vascular samples from myocardial sections and to selectively evaluate mRNA expressions of the individual tissue component.

Synergistic binding of DnaJ and DnaK chaperones to heat shock transcription factor σ32 ensures its characteristic high metabolic instability: implications for heat shock protein 70 (Hsp70)-Hsp40 mode of function.

Escherichia coli heat shock transcription factor σ(32) is rapidly degraded by ATP-dependent proteases, such as FtsH and ClpYQ. Although the DnaK chaperone system (DnaK, DnaJ, and GrpE) promotes σ(32) degradation in vivo, the precise mechanism that is involved remains unknown. Our previous results indicated that σ(32) mutants containing amino acid substitution in the N-terminal half of Region 2.1 are markedly stabilized in vivo. Here, we report the further characterization of these mutants by examining purified σ(32) mutants in vitro. Surprisingly, I54A σ(32), a very stable mutant, is more susceptible to ClpYQ and FtsH proteases than wild-type σ(32), indicating that the stability of σ(32) does not always reflect its susceptibility to proteases. Co-precipitation and gel filtration analyses show that purified σ(32) mutants exhibit a reduced affinity for DnaJ, leading to a marked decrease in forming a complex with DnaK in the presence of DnaJ and ATP. Other mutants with modestly increased stability (A50S σ(32) and K51E σ(32)) show an intermediate efficiency of complex formation with DnaK, suggesting that defects in binding to DnaK and DnaJ are well correlated with metabolic stability; effective interaction with DnaK promotes σ(32) degradation in vivo. We argue that the stable and effective interaction of heat shock protein 70 (Hsp70) with a substrate polypeptide may generally require the simultaneous binding of heat shock protein 40 (Hsp40) to distinct sites on the substrate.

Exaggerated blood pressure variability superimposed on hypertension aggravates cardiac remodeling in rats via angiotensin II system-mediated chronic inflammation.

Hypertensive patients with large blood pressure variability (BPV) have aggravated end-organ damage. However, the pathogenesis remains unknown. We investigated whether exaggerated BPV aggravates hypertensive cardiac remodeling and function by activating inflammation and angiotensin II-mediated mechanisms. A model of exaggerated BPV superimposed on chronic hypertension was created by performing bilateral sinoaortic denervation (SAD) in spontaneously hypertensive rats (SHRs). SAD increased BPV to a similar extent in Wistar Kyoto rats and SHRs without significant changes in mean blood pressure. SAD aggravated left ventricular and myocyte hypertrophy and myocardial fibrosis to a greater extent and impaired left ventricular systolic function in SHRs. SAD induced monocyte chemoattractant protein-1, transforming growth factor-beta, and angiotensinogen mRNA upregulations and macrophage infiltration of the heart in SHRs. The effects of SAD on cardiac remodeling and inflammation were much smaller in Wistar Kyoto rats compared with SHRs. Circulating levels of norepinephrine, the active form of renin, and inflammatory cytokines were not affected by SAD in Wistar Kyoto rats and SHRs. A subdepressor dose of candesartan abolished the SAD-induced left ventricular/myocyte hypertrophy, myocardial fibrosis, macrophage infiltration, and inductions of monocyte chemoattractant protein-1, transforming growth factor-beta, and angiotensinogen and subsequently prevented systolic dysfunction in SHRs with SAD. These findings suggest that exaggerated BPV induces chronic myocardial inflammation and thereby aggravates cardiac remodeling and systolic function in hypertensive hearts. The cardiac angiotensin II system may play a role in the pathogenesis of cardiac remodeling and dysfunction induced by a combination of hypertension and exaggerated BPV.

Mutant MCP-1 therapy inhibits tumor angiogenesis and growth of malignant melanoma in mice.

We investigated whether blocking of monocyte chemoattractant-1 (MCP-1) function would inhibit recruitment of tumor-associated macrophages (TAMs) and prevent tumor angiogenesis and tumor growth of human malignant melanoma. B16-F1 melanoma cells were implanted onto the back of C57BL/6 mice (Day 0). At Day 7, a dominant negative MCP-1 mutant (7ND) gene was transfected in the thigh muscle to make overexpressed 7ND protein secreted into systemic circulation. 7ND treatment inhibited TAM recruitment and partially reduced tumor angiogenesis and tumor growth. Also, 7ND treatment attenuated inductions of tumor necrosis factor-alpha (TNFalpha), interleukin-1alpha (IL-1alpha), and vascular endothelial growth factor (VEGF) in the stroma and tumor. Melanoma cells expressed not only MCP-1 but also its receptor CCR2. Accordingly, it was suggested that MCP-1 would enhance tumor angiogenesis and early tumor growth in the early stages by inducing TNFalpha, IL-1alpha, and VEGF through TAM recruitment and probably the direct autocrine/paracrine effects on melanoma cells.

Inhibition of intrinsic interferon-gamma function prevents neointima formation after balloon injury.

It is still controversial whether intrinsic interferon (IFN)-gamma promotes or attenuates vascular remodeling in hyperproliferative vascular disorders, such as neointima formation after balloon injury. Thus, we investigated whether inhibition of intrinsic IFN-gamma function prevents neointima formation. For this purpose, naked DNA plasmid encoding a soluble mutant of IFN-gamma receptor alpha-subunit (sIFNgammaR; an IFN-gamma inhibitory protein) or mock plasmid was injected into the thigh muscle of male Wistar rats 2 days before balloon injury (day -2). sIFNgammaR gene transfer significantly elevated serum levels of sIFNgammaR protein for 2 weeks. In mock-treated rats, balloon injury induced smooth muscle cell proliferation in the neointima with a peak at day 7 and produced thick neointima at day 14. sIFNgammaR treatment reduced the number of proliferating intimal smooth muscle cells by 50% at day 7 and attenuated neointima formation with a 45% reduction of the intima/media area ratio at day 14. In mock-treated rats, at day 7, balloon injury induced phosphorylation of signal transducer and activator of transcription-1 and upregulations of IFN regulatory factor-1 (a transcription factor mediating IFN-gamma signal). Balloon injury also upregulated the key molecules of neointima formation, such as intercellular adhesion molecule-1 and platelet-derived growth factor beta-receptor. These changes were suppressed by sIFNgammaR treatment. In conclusion, it is suggested that intrinsic IFN-gamma promotes neointima formation probably through IFN regulatory factor-1/intercellular adhesion molecule-1-mediated and platelet-derived growth factor-mediated mechanisms. Thus, inhibition of IFN-gamma signaling may be a new therapeutic target for prevention of neointima formation of hyperproliferative vascular disorders.

[A surgically treated case with a ruptured bacterial aneurysm of the middle cerebral arterial bifurcation following occlusion].

A 56-year-old woman with aortic regurgitation (AR) developd a high fever on April 25th, 2003, followed by the sudden onset of left hemiparesis and dysarthria on May 10th, 2003. MRI and MRA showed cerebral infarction due to occlusion of the right proximal portion of the middle cerebral artery. Streptococcus was isolated from arterial blood culture at the time of admission and cardiac examination such as echocardiography revealed active infective endocarditis. Cerebral angiography on the 31st day after the onset of symptoms demonstrated a fusiform-shaped aneurysm at the occluded M2 portion of the middle cerebral artery. Despite administration of antibiotics, a small subcortical hematoma was observed in the right temporal lobe surrounding the aneurysm on the 35th day. The direct surgery of aneurysmal trapping and resection was subsequently performed to prevent rebleeding. The sylvian fissure and perianeurysmal area were strongly adherent to granulation tissue and blood clot. After exposing the aneurysm, the dilated portion of the vessel was successfully trapped and resected. Other than residual left hemiparesis, the postoperative course was uneventful. Histological examination confirmed bacterial aneurysm due to bacterial embolization originating from infective endocarditis (IE). We report a rare case having a ruptured bacterial aneurysm of the middle cerebral arterial bifurcation requiring surgery following occlusion due to bacterial embolization after sepsis and meningitis due to infective endocarditis.