Pyogenic spondylitis with acute course caused by Corynebacterium simulans.

Corynebacterium simulans was first reported in 2000. Although it is a member of the normal skin flora, some cases of C. simulans infection have been reported. Other Corynebacterium spp. rarely cause chronic pyogenic spondylitis, and pyogenic spondylitis caused by C. simulans has not been reported at all. Here we report a case of acute pyogenic spondylitis due to C. simulans. A 78-year-old man with diabetes mellitus visited our hospital with a 3-day history of lower back pain and fever. Blood culture revealed C. simulans and magnetic resonance images of lumbar vertebrae showed pyogenic spondylitis. He recovered after treatment by vancomycin for 9 weeks and was discharged home. No recurrence has been observed for half a year. This is likely the first reported case of pyogenic spondylitis by C. simulans. In general, Corynebacterium spp. cause chronic pyogenic spondylitis, but this case showed an acute course.

Longitudinal changes in 18 F-THK5351 positron emission tomography in corticobasal syndrome.

BACKGROUND AND PURPOSE: Corticobasal syndrome (CBS) is pathologically characterized by tau deposits in neuronal and glial cells and by reactive astrogliosis. In several neurodegenerative disorders, 18 F-THK5351 has been observed to bind to reactive astrocytes expressing monoamine oxidase B. In this study, the aim was to investigate the progression of disease-related pathology in the brains of patients with CBS using positron emission tomography with 18 F-THK5351. METHODS: Baseline and 1-year follow-up imaging were acquired using magnetic resonance imaging and positron emission tomography with 18 F-THK5351 in 10 subjects: five patients with CBS and five age-matched normal controls (NCs). RESULTS: The 1-year follow-up scan images revealed that 18 F-THK5351 retention had significantly increased in the superior parietal gyrus of the patients with CBS compared with the NCs. The median increases in 18 F-THK5351 accumulation in the patients with CBS were 6.53% in the superior parietal gyrus, 4.34% in the precentral gyrus and 4.33% in the postcentral gyrus. In contrast, there was no significant increase in the regional 18 F-THK5351 retention in the NCs. CONCLUSIONS: Longitudinal increases in 18 F-THK5351 binding can be detected over a short interval in the cortical sites of patients with CBS. A monoamine oxidase B binding radiotracer could be useful in monitoring the progression of astrogliosis in CBS.

Control of mammary tumor differentiation by SKI-606 (bosutinib).

C-Src is infrequently mutated in human cancers but it mediates oncogenic signals of many activated growth factor receptors and thus remains a key target for cancer therapy. However, the broad function of Src in many cell types and processes requires evaluation of Src-targeted therapeutics within a normal developmental and immune-competent environment. In an effort to understand the appropriate clinical use of Src inhibitors, we tested an Src inhibitor, SKI-606 (bosutinib), in the MMTV-PyVmT transgenic mouse model of breast cancer. Tumor formation in this model is dependent on the presence of Src, but the necessity of Src kinase activity for tumor formation has not been determined. Furthermore, Src inhibitors have not been examined in an autochthonous tumor model that permits assessment of effects on different stages of tumor progression. Here we show that oral administration of SKI-606 inhibited the phosphorylation of Src in mammary tumors and caused a rapid decrease in the Ezh2 Polycomb group histone H3K27 methyltransferase and an increase in epithelial organization. SKI-606 prevented the appearance of palpable tumors in over 50% of the animals and stopped tumor growth in older animals with pre-existing tumors. These antitumor effects were accompanied by decreased cellular proliferation, altered tumor blood vessel organization and dramatically increased differentiation to lactational and epidermal cell fates. SKI-606 controls the development of mammary tumors by inducing differentiation.

Myeloablative hematopoietic stem cell transplantation for myelodysplastic syndrome in patients younger than 55 years: impact of comorbidity and disease burden on the long-term outcome.

We retrospectively investigated 31 myelodysplastic syndrome (MDS) patients receiving myeloablative hematopoietic stem cell transplantation (HCT) and focused on prognostic factors affecting the long-term outcome. Patients were classified according to the French-American-British classification and the HCT-comorbidity index was determined. Cytosine arabinoside or thiotepa combined with cyclophosphamide and total body irradiation was used as myeloablative conditioning in eight and 23 patients respectively. After a follow-up period of 0.8-14.2 years from transplantation (median: 6.4 years), 23 patients were alive in complete remission, and the 5-year overall survival (OS) and disease-free survival (DFS) rates were 79% and 72% respectively. The cumulative nonrelapse mortality (NRM) rate was 22% at 5 years. According to multivariate analysis, > or =20% blasts in the bone marrow and an HCT-comorbidity score > or = 3 were significantly associated with poor OS and DFS. Patients with a high HCT-comorbidity score and male patients receiving transplantation from female donors were significantly more likely to have a higher NRM according to the univariate, but not the multivariate analysis. These data suggest that comorbidity and the tumor burden at the time of transplantation may be useful variables for predicting the outcome in MDS patients receiving myeloablative HCT.

Modification of the hepatic mitochondrial proteome in response to ischemic preconditioning following ischemia-reperfusion injury of the rat liver.

BACKGROUND/AIM: Ischemic preconditioning (IPC) may reduce hepatic ischemia-reperfusion (IR) injury, but efficacy of IPC on mitochondrial proteome is not demonstrated. We investigated how IPC modifies the mitochondrial proteome after IR injury. METHODS: Rats were subjected to 25 min of portal triad crossclamping (IR group, n = 8). In the IPC group (n = 8), 10 min of temporal portal triad clamping was performed before 25 min of portal clamping. Samples were obtained after 24 h. The mitochondrial inner-membrane potential was measured by the uptake of a lipophilic cationic carbocyanine probe and mitochondrial proteome was also investigated using 2-dimensional differential in-gel electrophoresis and liquid chromatography-tandem mass spectrometry. RESULTS: Mitochondrial inner-membrane potential and glutathione were lower and serum transaminase was higher in the IPC group than in the IR group. The mitochondrial precursor of aldehyde dehydrogenase 2 and alpha-methylacyl-CoA-racemase were upregulated in the IPC group in comparison to the IR group. In contrast, protein disulfide-isomerase A3 precursor, 60S acid ribosomal protein P0, carbonic anhydrase 3 and superoxide dismutase were significantly more downregulated in the IPC group than in the IR group. CONCLUSIONS: A hepatoprotective effect by IPC was not shown; however, IPC caused significant up- or downregulation of several mitochondrial proteins.

Role of Gab2 in mammary tumorigenesis and metastasis.

Overexpression of the adaptor/scaffolding protein Gab2 has been detected in primary human breast cancer cells and cell lines, although its functional significance in breast carcinogenesis is not fully understood. Here, we show a requirement for Gab2 in promoting mammary tumor metastasis. Although Gab2 expression levels were elevated in mammary tumors induced by the Neu (ErbB-2) oncogene, homozygous deletion of Gab2 in mice had only a modest effect on the initiation of Neu-induced mammary tumors. Notably, ablation of Gab2 severely suppressed lung metastasis. Gab2-deficient cancer cells displayed normal Akt activities, and their proliferative rate in vitro was similar to control cells. However, Gab2(-/-) cancer cells exhibited decreased migration and impaired Erk activation, and the defects were rescued by re-introduction of Gab2 into Gab2(-/-) cells. These findings suggest that although Gab2 overexpression may confer growth advantage to tumor cells, the functional requirement for Gab2 in mammary tumor initiation/growth may be dispensable, and that Gab2 may have a prominent role in promoting mammary tumor metastasis.

Apoptosis and keratin intermediate filaments.

Intermediate filament (IF) proteins utilize central alpha-helical domains to generate polymeric fibers intermediate in size between actin microfilaments and microtubules. The regions flanking the central structural domains have diverged greatly to permit IF proteins to adopt specialized functions. Keratins represent the largest two groups of IF proteins. Most keratins serve structural functions in hair or epidermis. Intracellular epidermal keratins also provide strength to epithelial sheets. The intracellular type I keratins and other IF proteins are cleaved by caspases during apoptosis to ensure the disposal of the relatively insoluble cellular components. However, recent studies have also revealed an unexpected protective role for keratin 8 during TNF and Fas mediated apoptosis. Evidence for possible functions of keratins both upstream and downstream of apoptotic signaling are considered.

Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer.

Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.

Preparation of platinum nanoparticles by sonochemical reduction of the Pt(IV) ions: role of surfactants.

Sonochemical reduction processes of Pt(IV) ions in water have been investigated in the presence of various kinds of surfactants such as sodium dodecylsulfate (SDS) and sodium dodecylbenzenesulfonate (DBS) as anionic surfactants, and polyethylene glycol monostearate (PEG-MS) as non-ionic, dodecyltrimethylammonium chloride (DTAC) and bromide (DTAB) as cationic surfactants. An improved colorimetric determination reveals that Pt(IV) ion is reduced to zero valent metal in two steps: step (1)--Pt(IV) ion to Pt(II) ion, and step (2)--Pt(II) ion to Pt(0), and after the completion of step (1), step (2) sets in. It appears that rapid scrambling reactions among platinum ions and/or atoms, that is, Pt(I) + Pt(IV)-->Pt(II) + Pt(III), etc. take place. In the sonolysis of aqueous solutions of SDS, DBS or PEG-MS, two kinds of organic reducing radicals, R(ab) and R(py), are proposed to contribute to the reduction. Radical R(ab) is formed from the reaction of the surfactants with primary radicals such as hydroxyl radicals and hydrogen atoms originated from the sonolysis of water, and radical R(py) is formed from the direct thermal decomposition of surfactants in the interfacial region between the collapsing cavities and the bulk water. R(ab) is effective for both the reduction steps, whereas R(py) is involved only in the reduction step (1). This fact coincides with the previous reported sonochemical reduction of Pt(II) ions. Hydrogen atoms themselves scarcely participate in the reduction. The average diameter (1.0 nm) of platinum particles prepared from the system of PEG-MS is smaller than those from the aqueous solution of anionic surfactant SDS (3.0 nm) and DBS (3.0 nm).

Cytokeratins 8 and 19 in the mouse placental development.

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.

Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.

An Alu element from the K18 gene confers position-independent expression in transgenic mice.

We have identified a 323-base pair fragment of the 5'-flanking sequence of the K18 gene, which confers position-independent and copy number-dependent expression on two heterologous transgenes. This fragment is composed primarily of an Alu repetitive element. Its activity in mice is correlated with its RNA polymerase III promoter activity and its orientation-dependent ability to inhibit potential transcriptional interference in a transfection assay. However, the activity of the Alu element is not correlated with its enhancer blocking activity, a characteristic of insulator elements. In addition, this Alu element did not block the suppressive effect of co-injecting mouse alpha satellite DNA with the transgene. This Alu element is likely responsible for at least part of the protective effects of the sequences flanking the K18. These results suggest that transcriptionally active Alu elements may eliminate transcriptional interference of neighboring genes. This Alu element is one component of the locus control region associated with the K18 gene. Other Alu repetitive elements may also function to define regulatory domains.

A single targeted Ets2 allele restricts development of mammary tumors in transgenic mice.

Heterozygous female mice carrying a targeted mutation of the Ets2 transcription factor gene were mated with a mouse strain that develops mammary tumors due to the expression of the polyoma virus middle T oncogene. Tumors from females with only one wild-type Ets2 gene were approximately one-half the size of tumors from controls. The smaller size of the tumors was correlated with a more differentiated state of early hyperplastic growths and not to differential growth of the frank tumors or to decreased middle T gene expression. Ets2 may regulate the progression of these aggressive mammary tumors.

A regulatory element of the human keratin 18 gene with AP-1-dependent promoter activity.

The human keratin 18 (K18) gene is expressed in a restricted but diverse subset of differentiated epithelial tissues and carcinomas. The 10-kilobase pair K18 gene contains all of the genetic information necessary for tissue-specific, copy number-dependent and integration site-independent expression in transgenic mice. We identified a 100-base pair regulatory element that activates the K18 proximal promoter in the presence of the previously identified first intron enhancer. Deletion of the element greatly diminished K18 expression. This regulatory element also has cryptic, AP-1-dependent promoter activity in the absence of the normal promoter, which results in 10-40-fold higher levels of K18 RNA expression in transgenic mice. The high activity of this cryptic promoter is dependent upon the first intron enhancer. These experiments define interactive regulatory regions of the K18 gene that modulate expression in diverse epithelial cell types and identify an unusual regulatory element with promoter activity that may be useful for high level heterologous gene expression in transgenic animals.

A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells.

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys (mrr) gene. Direct protein-protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.

Defective trophoblast function in mice with a targeted mutation of Ets2.

Members of the Ets family of transcription factors mediate transcriptional responses of multiple signaling pathways in diverse cell types and organisms. Targeted deletion of the conserved DNA binding domain of the Ets2 transcription factor results in the retardation and death of homozygous mouse embryos before 8.5 days of embryonic development. Defects in extraembryonic tissue gene expression and function include deficient expression of matrix metalloproteinase-9 (MMP-9, gelatinase B), persistent extracellular matrix, and failure of ectoplacental cone proliferation. Mutant embryos were rescued by aggregation with tetraploid mouse embryos, which complement the developmental defects by providing functional extraembryonic tissues. Rescued Ets2-deficient mice are viable and fertile but have wavy hair, curly whiskers, and abnormal hair follicle shape and arrangement, resembling mice with mutations of the EGF receptor or its ligands. However, these mice are not deficient in the production of TGFalpha or the EGF receptor. Homozygous mutant cell lines respond mitogenically to TGFalpha, EGF, FGF1, and FGF2. However, FGF fails to induce MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in the Ets2-deficient fibroblasts. Ectopic expression of Ets2 in the deficient fibroblasts restores expression of both matrix metalloproteinases. Therefore, Ets2 is essential for placental function, mediating growth factor signaling to key target genes including MMP-3, MMP-9, and MMP-13 in different cell types, and for regulating hair development.

Probucol treatment attenuates the aortic atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

We examined the effect of probucol on the aortic atherosclerosis already developed in Watanabe heritable hyperlipidemic (WHHL) rabbits at the initiation of treatment. In WHHL rabbits treated with probucol for 5 months from 8 months old, the lesion area in the aorta was significantly (P < 0.05) reduced when compared with that in untreated animals as well as animals at age 8 months. In contrast, plasma cholesterol levels in the probucol-treated group and untreated group during the experiment were not significantly different. LDL prepared from rabbits receiving probucol for 5 months showed resistance to oxidation by copper ions. Plasma CETP activity was significantly (P < 0.05) increased by probucol treatment. An immunohistochemical study showed that macrophages were abundant in the atherosclerotic lesions of untreated rabbits whereas smooth muscle cells were predominant in lesions of probucol-treated rabbits. These results suggest that the atherosclerotic lesion in WHHL rabbits can regress when treated by probucol and that the attenuation of atherosclerosis in this animal involves effects of probucol other than a decrease in plasma cholesterol, for example anti-oxidant activity.

A regulatory element within a coding exon modulates keratin 18 gene expression in transgenic mice.

Multiple tissue-specific, DNase-hypersensitive sites are correlated with known or potential regulatory regions of the human keratin 18 (K18) gene. One of these sites is found within exon 6, close to a potential AP-1 binding site. Footprint analysis confirmed that this site is capable of binding c-Jun and c-Fos in vitro. However, exon 6 can stimulate expression of a reporter gene driven by the K18 proximal promoter independent of AP-1 in F9 cells and additionally modulates AP-1 responsiveness when in combination with an intron enhancer. Analysis in transgenic mice and by transient transfections of mutant forms of the K18 gene showed that exon 6 contributes to the expression of the K18 gene. However, substitution of part of exon 6 with the corresponding part of the keratin 19 gene which lacks an AP-1 site decreased but did not destroy the regulatory activity of the exon. Furthermore, this mutation did not alter either the tissue specificity or the position-independent and copy number-dependent behavior of the K18 gene. In contrast, a frameshift mutation within exon 6 dramatically decreased the expression of the gene. K18 RNA expression from the frameshift mutation was less than 10% of the wild type K18 transgene. This decline in expression was the result of a combination of decreased stability of mutant K18 RNA and the creation of a negative regulatory element that can interact with the first intron regulatory elements and actively suppress K18 expression. These results demonstrate that a protein-coding portion of the K18 gene also has a regulatory function.

Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression.

The activities of ETS transcription factors are modulated by posttranscriptional modifications and cooperation with other proteins. Another factor which could alter the regulation of genes by ETS transcription factors is DNA methylation of their cognate binding sites. The optimal activity of the keratin 18 (K18) gene is dependent upon an ETS binding site within an enhancer region located in the first intron. The methylation of the ETS binding site was correlated with the repression of the K18 gene in normal human tissues and in K18 transgenic mouse tissues. Neither recombinant ETS2 nor endogenous spleen ETS binding activities bound the methylated site effectively. Increased expression of the K18 gene in spleens of transgenic mice by use of an alternative, cryptic promoter 700 bp upstream of the enhancer resulted in modestly decreased methylation of the K18 ETS site and increased RNA expression. Expression in transgenic mice of a mutant K18 gene, which was still capable of activation by ETS factors but was no longer a substrate for DNA methylation of the ETS site, was fivefold higher in spleen and heart. However, expression in other organs such as liver and intestine was similar to that of the wild-type gene. This result suggests that DNA methylation of the K18 ETS site may be functionally important in the tissue-specific repression of the K18 gene. Epigenetic modification of the binding sites for some ETS transcription factors may result in a refractory transcriptional response even in the presence of necessary trans-acting activities.

Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis.

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light-induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.