Observation of quadrupole helix chirality and its domain structure in DyFe3(BO3)4.

Resonant X-ray diffraction (RXD) uses X-rays in the vicinity of a specific atomic absorption edge and is a powerful technique for studying symmetry breaking by motifs of various multipole moments, such as electric monopoles (charge), magnetic dipoles (spin) and electric quadrupoles (orbital). Using circularly polarized X-rays, this technique has been developed to verify symmetry breaking effects arising from chirality, the asymmetry of an object upon its mirroring. Chirality plays a crucial role in the emergence of functionalities such as optical rotatory power and multiferroicity. Here we apply spatially resolved RXD to reveal the helix chirality of Dy 4f electric quadrupole orientations and its domain structure in DyFe3(BO3)4, which shows a reversible phase transition into an enantiomorphic space-group pair. The present study provides evidence for a helix chiral motif of quadrupole moments developed in crystallographic helix chirality.

Neurogenic cardiomyopathy in rabbits with experimentally induced rabies.

Cardiomyopathies have been rarely described in rabbits. Here we report myocardial necrosis of the ventricular wall in rabbits with experimentally induced rabies. Myocardial lesions were found only in rabbits with brain lesions, and the severity of the cardiac lesions was proportional to that of the brain lesions. Neither the frequency nor the cumulative dose of anesthesia was related to the incidence or the severity of the myocardial lesions. The myocardial lesions were characterized by degeneration and/or necrosis of myocardial cells and were accompanied by contraction band necrosis, interstitial fibrosis, and infiltration of inflammatory cells. The brain lesions due to rabies virus infection were most prominent in the cerebral cortex, thalamus, hypothalamus, brainstem, and medulla. Rabies virus antigen was not found in the hearts of any rabbits. Based on these findings, the myocardial lesions were classified as neurogenic cardiomyopathy.

Flavivirus encephalitis: pathological aspects of mouse and other animal models.

Encephalitic flaviviruses are important arthropod-borne pathogens of humans and other animals. In particular, the recent emergence of the West Nile virus (WNV) and Japanese encephalitis virus (JEV) in new geographic areas has caused a considerable public health alert and international concern. Among the experimental in vivo models of WNV and JEV infection, mice and other laboratory rodents are the most thoroughly studied and well-characterized systems, having provided data that are important for understanding the infectious process in humans. Macaca monkeys have also been used as a model for WNV and JEV infection, mainly for the evaluation of vaccine efficacy, although a limited number of published studies have addressed pathomorphology. These animal models demonstrate the development of encephalitis with many similarities to the human disease; however, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterized.

Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.

bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

A novel red-emitting K2Ca(PO4)F:Eu2+ phosphor with a large Stokes shift.

We report a K2CaPO4F:Eu2+ phosphor with a new crystal structure. This phosphor has a large Stokes shift and converts near-ultraviolet light to red luminescence without absorption of other visible light. The mechanism was elucidated by applying a constrained density functional theory to the solved crystal structure.

First isolation of West Nile virus in Zambia from mosquitoes.

Mosquito surveillance studies to identify mosquito-borne flaviviruses have identified West Nile Virus (WNV) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.

Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS.

Signaling adaptor protein Crk regulates cell motility and growth through its targets Dock180 and C3G, those are the guanine-nucleotide exchange factors (GEFs) for small GTPases Rac and Rap, respectively. Recently, overexpression of Crk has been reported in various human cancers. To define the role for Crk in human cancer cells, Crk expression was targeted in the human ovarian cancer cell line MCAS through RNA interference, resulting in the establishment of three Crk knockdown cell lines. These cell lines exhibited disorganized actin fibers, reduced number of focal adhesions, and abolishment of lamellipodia formation. Decreased Rac activity was demonstrated by pull-down assay and FRET-based time-lapse microscopy, in association with suppression of both motility and invasion by phagokinetic track assay and transwell assay in these cells. Furthermore, Crk knockdown cells exhibited slow growth rates in culture and suppressed anchorage-dependent growth in soft agar. Tumor forming potential in nude mice was attenuated, and intraperitoneal dissemination was not observed when Crk knockdown cells were injected into the peritoneal cavity. These results suggest that the Crk is a key component of focal adhesion and involved in cell growth, invasion, and dissemination of human ovarian cancer cell line MCAS.

Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination.

Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Here we show by utilizing double-stranded RNA (dsRNA)-mediated genetic interference that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination in Caenorhabditis elegans. Injection of dsRNA into adult animals resulted in the inhibition of meiotic crossing over and induced the loss of chiasmata at diakinesis in oocytes of F(1) animals. However, electron microscopic analysis revealed that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appeared to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first example of Cds1-related kinases that are required for meiotic recombination in multicellular organisms.

Superconductivity of doped Ar@C60.

The synthesis of a mg amount of pure argon containing fullerene allowed the synthesis of the first endohedral superconductors with critical temperatures lower than expected, an indication of the strong influence of the argon atom on the C60 cage.

Alternatively spliced forms of cyclin D1 modulate entry into the cell cycle in an inverse manner.

Alternative splicing of cyclin D1 gene mRNA has recently been demonstrated. The novel transcript shows no splicing at the downstream exon 4 boundary and encodes a protein with an altered carboxyl-terminal domain that is a cyclin D1 variant; exon 5 is not included in the coding sequence which terminates downstream of exon 4. We here produced cells that exogenously express each form of cyclin D1 and analysed their cell cycle regulation. We found that (1) alternative splicing forms of cyclin D1 modulated entry into the cell cycle in an inverse manner; (2) both splicing forms suppressed cell growth; and (3) cells overexpressing form [a] were inhibited from entry into and completion of the S phase, although form [b]-expressing cells showed no reduction of G1- to S transition. We also found that overexpression of either cyclin D1 form upregulated Rb gene products, suggesting that this upregulation may be one of the causes of growth suppression in cyclin D1 overexpressing cells.

Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor.

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 x 10(-10) to 1 x 10(-6) M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 x 10(-6) M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10(-6) M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10(-10) - 10(-6) M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activity in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores.

Human cystine/glutamate transporter: cDNA cloning and upregulation by oxidative stress in glioma cells.

A human cDNA for amino acid transport system x(C)(-) was isolated from diethyl maleate-treated human glioma U87 cells. U87 cells expressed two variants of system x(C)(-) transporters hxCTa and hxCTb with altered C-terminus regions probably generated by the alternative splicing at 3'-ends. Both hxCTa and hxCTb messages were also detected in spinal cord, brain and pancreas, although the level of hxCTb expression appears to be lower than that of hxCTa in these tissues. When expressed in Xenopus oocytes, hxCTb required the heavy chain of 4F2 cell surface antigen (4F2hc) and exhibited the Na(+)-independent transport of L-cystine and L-glutamate, consistent with the properties of system x(C)(-). In agreement with this, 137 kDa band was detected by either anti-xCT or anti-4F2hc antibodies in the non-reducing condition in western blots, whereas it shifted to 50 kDa or 90 kDa bands in the reducing condition, indicating the association of two proteins via disulfide bands. We found that the expression of xCT was rapidly induced in U87 cells upon oxidative stress by diethyl maleate treatment, which was accompanied by the increase in the L-cystine uptake by U87 cells. Because of this highly regulated nature, xCT in glial cells would fulfill the task to protect neurons against oxidative stress by providing suitable amount of cystine to produce glutathione.

Angiotensin-converting enzyme inhibition attenuates hypofibrinolysis and reduces cardiac perivascular fibrosis in genetically obese diabetic mice.

BACKGROUND: Obesity and insulin resistance are associated with accelerated macrovascular and microvascular coronary disease, cardiomyopathic phenomena, and increased concentrations and activity in blood of plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of fibrinolysis. METHODS AND RESULTS: To determine whether hypofibrinolysis in blood and tissues and its potential sequelae could be attenuated pharmacologically, we studied genetically modified obese mice. By 10 weeks of age, obese mice exhibited increases in left ventricular weight and glucose and immunoreactive insulin in blood. PAI-1 activity in blood measured spectrophotometrically was significantly elevated as well. The difference compared with values in lean controls widened by 20 weeks of age. Perivascular fibrosis in coronary arterioles and small coronary arteries was evident in obese mice 10 and 20 weeks of age, paralleling increases in PAI-1 and tissue factor expression evident by immunohistochemical image analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. Inhibition of ACE activity initiated in obese mice 10 weeks of age and continued for 20 weeks arrested the increase in PAI-1 activity in blood and in cardiac PAI-1 and tissue factor mRNA as well as coronary perivascular fibrosis. CONCLUSIONS: Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.

Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: a potential progenitor of restenosis.

OBJECTIVES: This study was performed to determine whether altered gene expression of plasminogen activator inhibitor type 1 (PAI-1) occurs within the arterial wall after experimentally induced balloon injury. BACKGROUND: PAI-1, known to inhibit fibrinolysis in the circulation and to be present within atherosclerotic vessels, may influence proteolysis in the arterial wall and neointimal formation after angioplasty. METHODS: In rabbit carotid arteries subjected to balloon injury, both PAI-1 gene and protein expression were assayed sequentially with the use of Northern blotting, in situ hybridization and immunohistochemical studies. RESULTS: In uninjured, normal vessels PAI-1 messenger ribonucleic acid (mRNA) was not detectable by Northern blotting or in situ hybridization. However, injury was followed within 3 h by increases in PAI-1 mRNA (3.2 kb) of 5.9-fold compared with that in contralateral control carotid arteries (Northern blots). PAI-1 mRNA was detectable by in situ hybridization early after injury first in adventitia; after 24 h it was particularly prominent in the media. From 1 to 4 weeks after injury it was consistently detectable and was localized in neointimal vascular smooth muscle and endothelial cells at a time when neointimal thickening was marked. Cells of both types exhibited PAI-1 protein detected immunohistochemically. In vessels maintained in organ culture after balloon injury in vivo, sustained increases in PAI-1 activity appeared in conditioned media as well. CONCLUSIONS: Our results indicate that balloon injury simulating angioplasty in patients induces intramural expression of PAI-1 in vascular smooth muscle and endothelial cells. The decreased cell surface fibrinolytic activity likely to result from the increased PAI-1 expression may initiate or exacerbate mural thrombosis. Accordingly, excessive stimulation with clot-associated mitogens may stimulate vascular smooth muscle cell proliferation, which, coupled with increased accumulation of extracellular matrix attributable to decreased plasmin-mediated degradation, may contribute to restenosis.

Serious cardiac and pulmonary calcification in a young peritoneal dialysis patient: potential role of continuous correction of acidosis.

We describe a 40-month-old male infant with renal failure, treated with peritoneal dialysis, who developed massive calcification of soft tissues including the heart and lungs with subsequent cardiopulmonary insufficiency. A diagnosis of Jeune syndrome was made. After starting peritoneal dialysis, the patient exhibited an intractable metabolic acidosis of unknown etiology necessitating treatment with intravenous or oral sodium bicarbonate. Myocardial calcification was first detected by 2-dimensional echocardiography performed 3 months after starting dialysis. The patient was not suitable for renal transplantation because of his cardiac dysfunction and died of cardiac and respiratory failure at the age of 6 years. Although the patient exhibited a variety of risk factors for ectopic calcification including hyperphosphatemia, hyperparathyroidism, high calcium-phosphate product and treatment with vitamin D, the early and massive soft tissue calcification may have been accelerated by correction of the metabolic acidosis. Therefore, the use of sodium bicarbonate may be involved in the etiology of the myocardial calcification.

Cloning and characterization of a rat ortholog of MMP-23 (matrix metalloproteinase-23), a unique type of membrane-anchored matrix metalloproteinase and conditioned switching of its expression during the ovarian follicular development.

In our attempt to study the role of matrix metalloproteinases (MMPs) in the process of mammalian ovulation, we isolated a rat ortholog of the recently reported human MMP-23 from gonadotropin-primed immature rat ovaries. Transient expression of epitope-tagged rat and human MMP-23 in COS-1 cells revealed that they were synthesized as a membrane-anchored glycoprotein with type II topology. Indirect immunofluorescent analysis showed that subcellular localization of MMP-23 was predominantly in the perinuclear regions. The transfected human MMP-23 protein was processed endogenously to the soluble form in COS-1 cells. However, cotransfection of MMP-23 with the mouse furin cDNA did not enhance this processing, indicating that furin may not be involved in this event. Notably, in situ hybridization analysis revealed a dramatic switching of MMP-23 mRNA localization from granulosa cells to theca-externa/fibroblasts and ovarian surface epithelium during the follicular development. In serum-free primary culture of rat granulosa cells, a drastic diminution of MMP-23 mRNA expression was observed in response to FSH action between 24 h and 48 h of culture. The observed effect of FSH on MMP-23 expression was mimicked by treatment of granulosa cells with forskolin or 8-bromo (Br)-cAMP. In contrast, MMP-23 mRNA levels increased in theca-interstitial cells regardless of the presence of LH in the culture. However, treatment of theca-interstitial cells with forskolin or 8-Br-cAMP markedly reduced the expression of MMP-23 with a concomitant increase in progesterone production. These results indicate that the MMP-23 gene is spatially and temporally regulated in a cell type-specific manner in ovary via the cAMP signaling pathway.

Surface proteins localized in ruffling membranes and filopodia of human glioma cells detected using Mab S-11E10.

We prepared mouse monoclonal antibody (Mab S-11E10) for the surface antigen specifically distributed in ruffling membranes and filopodia of cultured human glioma cells. The antibody reacted to the specified structures of other glioma cells (U251MG, KNS 60) as well as those of KNS 42 cells, which were the immunizing source. The antigens, identified by Mab S-11E10, had molecular weights of 65 and 66 kDa. Immunohistochemically, the antibody reacted only to astrocytes in the human brain, but the cross-reactivity was noted in extraneural tissues such as lymphocytes and epithelium of the bowel ducts. In 5 out of 6 low grade astrocytomas, most of the tumor cells were strongly stained, while tumor cells of highly cellular areas of anaplastic astrocytomas and glioblastomas were either not stained or only weakly stained. The specific localization of 65-66 kDa doublet proteins may suggest relation to spreading and locomotion of cells, and may correlate to astrocytic differentiation and/or function.

Infection with JC virus and possible dysplastic ganglion-like transformation of the cerebral cortical neurons in a case of progressive multifocal leukoencephalopathy.

Infection of the cerebral cortical neurons with JC virus (JCV) with possible dysplastic ganglion-like alteration of the infected neurons found in a case of progressive multifocal leukoencephalopathy (PML) is described. The patient was a 21-year-old man with common variable immunodeficiency who died of PML after a 9-month clinical course. At autopsy, the white matter of the cerebrum, brainstem, cerebellum, and spinal cord exhibited extensive demyelination and necrosis. Numerous inclusion-bearing oligodendrocytes and bizarre astrocytes were found. In the occipital and temporal cortex, thick band-like aggregates of dysplastic ganglion-like cells (DGLCs) were found. These DGLCs showed immunohistochemical properties of neurons, and nuclei of some DGLCs were immunoreactive for large T antigen of SV40/JCV and p53, but not for capsid protein JCV VP1. In situ hybridization for mRNA of JCV large T antigen revealed positive signals in the nuclei of some DGLCs. These results indicate that JCV infected neurons and it is suggested that binding of the large T antigen with cellular proteins could have resulted in the dysplastic, ganglion cell-like change of the infected neurons, although the possibility that the aggregates of DGLCs represent a pre-existent malformative lesion of the cortex cannot be excluded completely.

Expression of beta 1 integrins in human endometrial stromal and decidual cells.

The present study was undertaken to investigate the expression of beta1 integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the beta 1, alpha 1, alpha 2, and alpha 5 subunits of the beta1 integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of beta1 integrins in vitro. The immunohistochemical distribution of beta1 integrins demonstrated predominantly glandular epithelial staining in the proliferative phase, and mesenchymal and glandular staining in the midsecretory phase. Flow cytometry also demonstrated the expression of the beta 1, alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of beta 1 integrin family in cultured decidual cells, and the enriched-fraction of prolactin (PRL)-producing decidual cells isolated by Percoll gradients showed high levels of beta 1, integrins expression. Immunohistochemistry confirmed the beta 1 integrin cell surface phenotypes in cultured decidual cells observed by flow cytometry. Autoradiography of immunoprecipitate subjects to SDS-PAGE revealed three major polypeptides with molecular weights of 130 kDa (beta 1 subunit), 165 kDa (alpha 2 subunit), and 210 kDa (alpha 1 subunit) under reducing conditions. In summary, the present study demonstrated that endometrial stromal and decidual cells expressed beta1 integrin subunits at their surfaces. The expression exhibited a variability throughout the menstrual cycles, being predominantly detected in the secretory phase, and was maintained highly in the decidua. Thus, beta 1 integrins in human endometrium and decidua may be important in mediating the organization of extracellular matrix proteins derived from embryos during the early stage of implantation.