Isolation and radiation hybrid mapping of dinucleotide repeat polymorphism at the human matrix Gla protein (MGP) locus

Matrix Gla protein (MGP) is an 84-residue, vitamin K-dependent protein expressed by chondrocytes and vascular smooth muscle cells, and is a potent regulator of calcium deposition in cartilage and arterial wall. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human MGP gene. This polymorphism will be useful in genetic studies of arteriosclerosis and osteoporosis. Received: November 5, 1997 / Accepted November 27, 1997     

Electron Microscopic Studies on IgA Nephropathy

We carried out electron microscopic studies on renal tissues from 9 patients with IgA nephropathy. Electron dense deposits were present in the mesangial area in all cases, subendothelial deposits in 4, and subepithelial deposits in only one. In basement membrane, segmental swelling and rarefaction of basement membrane substance were observed. In some cases the degenerated basement membrane substance protruded through the dilated endothelial fenestration into capillary lumina. Focal splitting, attenuation, mouse eaten appearance, and herniation of basement membrane were seen in a high incidence. Mesangial cells possessed well developed rough endoplasmic reticulums and polysomes. In the peripheral areas of mesangial cell cytoplasm, there was accumulation of electron dense substance and this was occasionally continuously present in the mesangial matrix. There was segmental swelling of endothelial cell cytoplasm, resulting in loss of fenestration. Epithelial cells had well developed rough endoplasmic reticulums and polysomes. Segmental foot process fusion was seen, and these processes, regardless of fusion, became electron denser in the area close to the basement membrane. Multivesiculated bodies were present in all cases in the epithelial cells and in 7 cases in the endothelial cells. Spherical microparticles were present in 3 cases in the urinary space or between the basement membrane and the epithelial cells.     

Reduced-intensity unrelated cord blood transplantation for patients with advanced malignant lymphoma.

We report the results of reduced-intensity unrelated cord blood transplantation (RI-UCBT) in patients with advanced malignant lymphoma. Twenty patients (median age, 46.5 years; range, 27-66 years) underwent RI-UCBT with a preparative regimen consisting of fludarabine 125 mg/m2 , melphalan 80 mg/m 2 , and 4 Gy of total body irradiation. The median infused total cell dose was 2.75 x 10(7)/kg (range, 2.3-3.4 x 10(7)/kg). Graft-versus-host disease (GVHD) prophylaxis was composed of cyclosporine or tacrolimus alone. Fifteen patients achieved primary neutrophil engraftment after a median of 20 days. Eight patients developed grade II to IV acute GVHD, and 2 developed chronic GVHD. Of the 16 patients with evaluable disease, 10 achieved a complete response. Primary disease recurred in 1 patient, and transplant-related mortality within 100 days occurred in 8 of 20 patients. The estimated 1-year probability of progression-free survival was 50%. These data suggest that RI-UCBT is a feasible option for patients with refractory lymphoma who lack an HLA-matched donor.     

Contribution of redox-active iron and copper to oxidative damage in Alzheimer disease

Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury, affecting every category of macromolecule, and are central to the oxidative injury hypothesis of Alzheimer disease (AD) pathogenesis. Studies on redox-competent copper and iron indicate that redox activity in AD resides exclusively within the neuronal cytosol and that chelation with deferoxamine, DTPA, or, more recently, iodochlorhydroxyquin, removes this activity. We have also found that while proteins that accumulate in AD possess metal-binding sites, metal-associated cellular redox activity is primarily dependent on metals associated with nucleic acid, specifically cytoplasmic RNA. These findings indicate aberrations in iron homeostasis that, we suspect, arise primarily from heme, since heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in AD, and mitochondria, since mitochondria turnover, mitochondrial DNA, and cytochrome C oxidative activity are all increased in AD. These findings, as well as studies demonstrating a reduction in microtubule density in AD neurons, suggest that mitochondrial dysfunction, acting in concert with cytoskeletal pathology, serves to increase redox-active heavy metals and initiates a cascade of abnormal events culminating in AD pathology.     

Advances in neuroimmunological laboratory studies on neuromuscular diseases

The recent methodological advances in molecular biology, immunology, and genetics have clarified neuroimmunological problems in axonal Guillain-Barré syndrome, seronegative myasthenia gravis, paraneoplastic neurologic syndromes and many others. In addition to clinical and serological studies in peripheral neuropathies, the origins and measurement of anti-ganglioside antibodies and relationships to similar carbohydrate structures on infectious organisms are discussed in the context of molecular mimicry hypothesis, especially related with both the localization of relevant gangliosides in the nerve and clinical phenotypes. Major advances have been made in animal modeling of anti-ganglioside antibody-associated disease. An explanation for muscular weakness in 10-15% of patients with seronegative myasthenia gravis who lack autoantibodies to acetylcholine receptors(AchRs) appears to be the autoantibodies to muscle-specific receptor tyrosine kinase (MuSK). MuSK mediates agrin-induced clustering of AchRs during synapse formation. These autoantibodies to the extracellular domain of MuSK inhibit its function in tissue culture. Isoelectric focusing (IEF) and agar gel electrophoresis (AGE) are used to examine cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis (MS). The CSF oligoclonal IgG bands (OB) are less frequently observed in Japanese MS patients compared with Caucasian patients. Few optic-spinal form of MS (OS-MS) was positive for OB by agarose gel electrophoresis, but IEF is more sensitive than AGE. Recent IEF data revealed some OS-MS patients were positive for OB. The neuroimmunological advances revealed the relationship between the neuroimmunological diseases and the putative autoantibodies as diagnostic markers, for example, HAM and hnRNP-A1, MSand anti-hnRNP-B1 antibody, opsoclonus-myoclonus syndrome and anti-GluRdelta2 antibody, Rasmussen encephalitis and anti-GluR3 antibody, paraneoplastic brainstem encephalitis and anti-Ma2 antibody, and so on.     

Development of the articular cavity in the rat temporomandibular joint with special reference to the behavior of endothelial cells and macrophages

Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively.     

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable,epigenetic gene silencing: a mammalian cell culture model of gene variegation

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.     

Intercellular adhesion molecule-1 in patients with idiopathic interstitial pneumonia

This study focuses on a possible role of intercellular adhesion molecule-1 (ICAM-1) in interstitial pulmonary diseases. We determined a soluble form of ICAM-1 in serum and bronchoalveolar lavage fluid (BALF) using ELISA in patients with usual interstitial pneumonia (UIP), bronchiolitis obliterance organizing pneumonia (BOOP), or nonspecific interstitial pneumonia (NSIP). In addition, we investigated the expression of ICAM-1 in the lung tissues of these patients by means of immunohistochemical staining. Serum levels of soluble ICAM-1 were significantly higher in patients with UIP or NSIP than in healthy subjects, and were also high in patients with BOOP. The soluble ICAM-1 in BALF tended to be higher in patients with UIP, BOOP, or NSIP than in normal subjects. A significant correlation was seen between soluble levels of ICAM-1 in serum and BALF. In the immunostaining of ICAM-1 of the lung tissues, ICAM-1 expression was more pronounced in patients with UIP than in those with BOOP or NSIP. The increased expression of ICAM-1 was seen in type II alveolar epithelium and vascular endothelium in patients with interstitial pneumonia. A positive correlation was observed between the degree of ICAM-1 expression in the lung tissues and the BALF levels of soluble ICAM-1. The expression of ICAM-1 in type II alveolar epithelium suggests that ICAM-1 plays a specific role in the fibrotic process of the lung, and that the measurement of soluble ICAM-1 in sera and BALF could be a useful marker for evaluating the progression of fibrosis.     

Regulation by Ca2+-calmodulin of the actin-bundling activity ofPhysarum 210-kDa protein

Summary From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca²⁺. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 ± 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 Å and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.     

Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis

Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.