Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice.

OBJECTIVE: Moderate mechanical stress generated by normal joint loading and movement is essential for the maintenance of healthy articular cartilage. However, the effects of reduced loading caused by the absence of weight bearing or joint motion on articular cartilage and subchondral bone is still poorly understood. We aimed to characterize morphological and metabolic responses of articular cartilage and subchondral bone to decreased mechanical stress in vivo. METHODS: Mice were subjected to periods of hindlimb unloading by tail suspension or external fixation of the knee joints. The articular surface was observed with digital microscope and the epiphyseal bone was assessed by micro-CT analysis. Articular cartilage and subchondral bone were further evaluated by histomorphometric, histochemical, and immunohistochemical analyses. RESULTS: The joint surface was intact, but thickness of both the total and uncalcified layer of articular cartilage were decreased both after joint unloading and immobilization. Subchondral bone atrophy with concomitant marrow expansion predisposed osteoclast activity at bone surface to invade into cartilaginous layer. Uncalcified cartilage showed decreased aggrecan content and increased aggrecanase expression. Alkaline phosphatase (ALP) activity was increased at uncalcified cartilage, whereas decreased at calcified cartilage. The distributions of hypertrophic chondrocyte markers remained unchanged. CONCLUSION: Thinning of articular cartilage induced by mechanical unloading may be mediated by metabolic changes in chondrocytes, including accelerated aggrecan catabolism and exquisitely modulated matrix mineralization, and cartilage matrix degradation and resorption by subchondral osteoclasts. Cartilage degeneration without chondrocyte hypertrophy under unloading condition indicate the possible existence of mechanism which is different from osteoarthritis pathogenesis.

Denervation-Associated Change in the Palatinus and Levator Veli Palatini Muscles of Dogs with Elongated Soft Palate.

Muscle lesions and decreased numbers of peripheral nerve branches have been reported in the soft palates of dogs presenting with brachycephalic airway obstruction syndrome (BAOS). Myosin adenosine triphosphatase staining was employed to investigate whether muscle lesions in the elongated soft palate (ESP) of dogs with BAOS reflect the presence of denervation. Soft palates were collected from nine brachycephalic dogs during surgical intervention for BAOS and from five healthy beagle dogs as controls. In the control soft palates, myofibres with relatively uniform diameters and a random mosaic pattern of type I and II myofibres were observed in the palatinus muscle (PM), while almost all of the myofibres in the levator veli palatini muscle (LVPM) were of type II. In the ESPs, small group atrophy, large group atrophy and angular-shaped atrophy were observed in myofibres of the PM and rarely in the LVPM. Fibre type grouping and an increase in type IIC myofibres were found only in the PM. Morphometric analysis of ESPs revealed a significant increase in the number of type I and II myofibres in the PM showing atrophy or hypertrophy compared with controls. A significant increase in atrophic type II myofibres was found in the LVPM of affected dogs. Myopathy consistent with denervation was observed in the PM, but rarely in the LVPM, of ESP specimens. The results suggest that the myopathy seen in dogs with ESP may partly reflect atrophy of myofibres resulting from damage to peripheral nerve branches, with subsequent reinnervation of myofibres.

Bisulfite sequencing and dinucleotide content analysis of 15 imprinted mouse differentially methylated regions (DMRs): paternally methylated DMRs contain less CpGs than maternally methylated DMRs.

Imprinted genes in mammals show monoallelic expression dependent on parental origin and are often associated with differentially methylated regions (DMRs). There are two classes of DMR: primary DMRs acquire gamete-specific methylation in either spermatogenesis or oogenesis and maintain the allelic methylation differences throughout development; secondary DMRs establish differential methylation patterns after fertilization. Targeted disruption of some primary DMRs showed that they dictate the allelic expression of nearby imprinted genes and the establishment of the allelic methylation of secondary DMRs. However, how primary DMRs are recognized by the imprinting machinery is unknown. As a step toward elucidating the sequence features of the primary DMRs, we have determined the extents and boundaries of 15 primary mouse DMRs (including 12 maternally methylated and three paternally methylated DMRs) in 12.5-dpc embryos by bisulfite sequencing. We found that the average size of the DMRs was 3.2 kb and that their average G+C content was 54%. Dinucleotide content analysis of the DMR sequences revealed that, although they are generally CpG rich, the paternally methylated DMRs contain less CpGs than the maternally methylated DMRs. Our findings provide a basis for the further characterization of DMRs.

cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans.

A high frequency of apoptosis is a conserved hallmark of oocyte development. In C. elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors. We have investigated the functions of CGH-1, the C. elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast. We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles. cgh-1 is required for oocyte and sperm function. It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism. We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C. elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species.

The Caenorhabditis elegans unc-32 gene encodes alternative forms of a vacuolar ATPase a subunit.

Eukaryotes possess multiple isoforms of the a subunit of the V(0) complex of vacuolar-type H(+)-ATPases (V-ATPases). Mutations in the V-ATPase a3 isoform have recently been shown to result in osteopetrosis, a fatal disease in humans, but no function has yet been ascribed to other isoforms. In Caenorhabditis elegans, the unc-32 mutant was originally isolated on the basis of its movement defect. We have isolated four new mutant alleles, the strongest of which is embryonic lethal. We show here that unc-32 corresponds to one of the four genes encoding a V-ATPase a subunit in the nematode, and we present their expression patterns and a molecular analysis of the gene family. unc-32 gives rise via alternative splicing to at least six transcripts. In the uncoordinated alleles, the transcript unc-32 B is affected, suggesting that it encodes an isoform that is targeted to synaptic vesicles of cholinergic neurons, where it would control neurotransmitter uptake or release. Other isoforms expressed widely during embryogenesis are mutated in the lethal alleles and would be involved in other acidic organelles. Our results indicate that V-ATPase a subunit genes are highly regulated and have tissue-specific function.

[A case of Fabry's disease with chronic renal failure].

Fabry's disease is a genetic disorder caused by the absence of alpha-galactosidase (alpha-Gal), the gene of which is carried on the long arm of the X chromosome. This enzymatic defect leads to an accumulation of glycosphingolipids in the plasma and lysosomes of endothelial, perithelial, and smooth muscle cells, especially involving those of the cardiovascular, renal and cerebrovascular systems. We report one male case of Fabry's disease with renal deterioration. A 36-year-old man who was a classic case with acroparesthesia, angiokeratoma, and hypohidrosis from 10 years of age, was diagnosed to be a hemizygote of Fabry's disease at 27 years as a result of severe decreased alpha-Gal activity of his peripheral white blood cells. This patient was found to have a point mutation of a G to A transition in exon 1. In May, 1989, he was reported to have proteinuria with normal renal function and admitted to our hospital due to renal deterioration in September, 1993. Laboratory examinations revealed a serum urea nitrogen of 65 mg/dl and creatinine value of 6.9 mg/dl. Urinary protein excretion was 3.9 g/day and urinary sugar was negative. On the renal biopsy specimens, light microscopic examinations revealed multiple sclerosing and collaptic lesions in glomeruli without severe tubulo-interstitial damage, but with stenotic change of the small arteries and arterioles. Electron microscopic examinations revealed a large number of electron dense deposits in the tubules. We diagnosed this case as Fabry's disease with chronic renal failure, however the pathogenesis of this renal progressive deterioration remained obscure. In this case, degenerative changes in the renal vessels due to Fabry's disease may be associated with rapid deterioration in renal function.

Hairy cell leukemia with translocation (11;20)(q13;q11) and overexpression of cyclin D1.

We report on a male Japanese patient with hairy cell leukemia (HCL). A cytogenetic study with lipopolysaccharide stimuli showed a novel translocation (11;20)(q13;q11) in 10% of the analyzed cells. Northern blot analysis and RT-PCR analysis for cyclin D1 revealed the overexpression of cyclin D1, although the southern blot analysis of PRAD1 gene showed no rearrangement. In this particular case, the t(11;20)(q13;q11) might play some role in the oncogenesis of HCL and the overexpression of cyclin D1.

Liver cirrhosis with marked thrombocytopenia and highly elevated serum thrombopoietin levels.

Three patients with liver cirrhosis (LC) and a bleeding tendency due to marked thrombocytopenia of less than 20 x 10(9)/l were admitted to our hospital for further examination. Bone marrow examination revealed megakaryocytic hypoplasia in all three patients. All patients exhibited amegakaryocytic thrombocytopenic purpura, myelodysplastic syndrome, or bone marrow hypoplasia. 111In-labeled platelet kinetic studies revealed decreased platelet production in all patients. Although serum thrombopoietin (sTPO) levels are usually within the normal level in patients with LC, the sTPO levels of our patients were about 10 times higher than the levels of normal subjects (1.22 +/- 0.37 fmol/ml): 13.34, 16.79, and 10.46 fmol/ml, respectively. These sTPO data supported our findings of decreased megakaryopoiesis. Our findings suggest that examination of sTPO levels is useful in determining the etiology of marked thrombocytopenia in LC patients.

Polygenic control of the expression of biological activities of an antitumor polysaccharide, lentinan.

Genetic studies were carried out on two in vivo responses of lentinan, delayed type-acute phase responses (DT-APR) and vascular dilation and hemorrhage (VDH). Linkage analyses showed that DT-APR was controlled by two recessive genes, ltnr1 and ltnr2, which were mapped on chromosome 3 and 11, respectively. VDH was also found to be controlled by polygenes. One dominant major gene, Ltnr3, and three dominant minor genes, Ltnr4, Ltnr5, and Ltnr6, were mapped on chromosomes 6, 9, 15 and 16, respectively.

Two genes controlling acute phase responses by the antitumor polysacch aride, lentinan.

Lentinan, a beta-1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) and Mus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence length polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 and ltn1.2) responsible for DT-APR. ltn1.1 is closely linked to D3Mit11 on chromosome 3 and ltn1.2 to D11Nds9 on chromosome 11 (P <0.001). The linkage analysis also suggested that ltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that the ltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.

[Giant left atrium in mitral valve disease: a new plication procedure to relieve the compressions of left ventricular wall left bronchus and right lung].

The giant left atrium associated with mitral valve disease frequently produces postoperative hazardness in relation to hemodynamic and respiratory management. We have defined the most serious disorders induced by the presence of giant left atrium in three categories as follows. First, hemodynamic disturbance by the compression of left ventricular wall by downward extension of left atrium (type I), secondly, respiratory disturbance yielded by the compression of left main bronchus by upward extension of left atrium (type II), thirdly, compression of right middle lobe by rightward extension of left atrium (type III). A new procedure of para-annular, superior and right-side plication methods were derived as the procedure to relieve those compressions induced by giant left atrium. Up to the present, 47 patients with giant left atrium underwent surgery, twelve of valvular procedure only and thirty-five of valvular as well as plication procedure. The incidence of postoperative low output syndrome and respiratory failure were evaluated. The plication procedure showed marked decrease in the incidence of low output syndrome and respiratory failure postoperatively, eventual significant decrease in mortality rate. We conclude that plication procedure is very effective for the treatment of compression in the presence of giant left. atrium.