Lzts1 controls both neuronal delamination and outer radial glial-like cell generation during mammalian cerebral development.

In the developing central nervous system, cell departure from the apical surface is the initial and fundamental step to form the 3D, organized architecture. Both delamination of differentiating cells and repositioning of progenitors to generate outer radial glial cells (oRGs) contribute to mammalian neocortical expansion; however, a comprehensive understanding of their mechanisms is lacking. Here, we demonstrate that Lzts1, a molecule associated with microtubule components, promotes both cell departure events. In neuronally committed cells, Lzts1 functions in apical delamination by altering apical junctional organization. In apical RGs (aRGs), Lzts1 expression is variable, depending on Hes1 expression levels. According to its differential levels, Lzts1 induces diverse RG behaviors: planar division, oblique divisions of aRGs that generate oRGs, and their mitotic somal translocation. Loss-of-function of lzts1 impairs all these cell departure processes. Thus, Lzts1 functions as a master modulator of cellular dynamics, contributing to increasing complexity of the cerebral architecture during evolution.

High levels of DNA polymerase ß mRNA corresponding with the high activity in Graves' thyroid tissue.

INTRODUCTION: High DNA polymerase ß activity has been observed in the thyroid tissue of patients with Graves' disease (Nagasaka et al. in Metabolism 37:1051-1054, 1988). This fact aroused our interest in whether the alteration of DNA polymerase ß activity depends on DNA polymerase ß (DNA poly ß) mRNA levels, which may be modulated by thyroid-stimulating hormone (TSH) or thyroid-stimulating substances, i.e. TSH receptor antibody (TRAb). RESULT: Addition of TSH or TRAb to primary cultures of Graves' disease thyroid cells for 4 h led to no increase in DNA poly ß mRNA levels. In contrast, thyroid hormone synthesizing enzyme, peroxidase, mRNA levels increased fivefold after coculture with TSH and TRAb, even though DNA poly ß activity and mRNA levels are already significantly higher in Graves' disease thyroid tissues, compared with normal thyroid tissue. DISCUSSION: These results indicate that DNA poly ß expression in Graves' disease thyroid cells may be maximally activated or plateau in response to thyroid-stimulating immunoglobulins, or that the activation of to poly ß expression may occur via pathways other than the G protein and cyclic AMP system.

Apaf-1-independent programmed cell death in mouse development.

Many cells die during mammalian development and are engulfed by macrophages. In DNase II(-/-) embryos, the TUNEL-positive DNA of apoptotic cells is left undigested in macrophages, providing a system for studying programmed cell death during mouse development. Here, we showed that an Apaf-1-null mutation in the DNase II(-/-) embryos greatly reduced the number of macrophages carrying DNA at E11.5. However, at later stages of the embryogenesis, a significant number of macrophages carrying undigested DNA were present in Apaf-1(-/-) embryos, indicating that cells died and were engulfed in an Apaf-1-independent manner. In most tissues of the Apaf-1(-/-) embryos, no processed caspase-3 was detected, and the DNA of dead cells accumulated in the macrophages appeared intact. Many nonapoptotic dead cells were found in the tail of the Apaf-1(-/-) embryos, suggesting that the Apaf-1-independent programmed cell death occurred, and these dead cells were engulfed by macrophages. In contrast, active caspase-3 was detected in E14.5 thymus of Apaf-1(-/-) embryos. Treatment of fetal thymocytes with staurosporine, but not etoposide, induced processing of procaspases 3 and 9, indicating that the E14.5 thymocytes have the ability to undergo caspase-dependent apoptosis in an Apaf-1-independent manner. Thus, programmed cell death in mouse development, which normally proceeds in an efficient Apaf-1-depenent mechanism, appears to be backed up by Apaf-1-independent death systems.

CD4+CD25high regulatory T cells are markedly decreased in blood of patients with pemphigus vulgaris.

BACKGROUND: It remains to be determined whether pemphigus vulgaris (PV), an autoimmune blistering disease, has a reduction and/or dysfunction of CD4(+)CD25(high) regulatory T (Treg) cells. OBJECTIVES: To evaluate the frequency and phenotypes of Treg cells in blood of patients with PV. METHODS: Peripheral blood mononuclear cells were prepared from PV patients as well as normal and disease control volunteers, and the frequency and phenotypes of Treg cells were determined by flow cytometry. CD4(+)CD25(+) and CD4(+)CD25(-) T cells isolated from peripheral blood mononuclear cells of PV patients and normal controls were subjected to real-time semiquantitative RT-PCR for the expression of Foxp3 gene. RESULTS: The proportion of Treg cells in all PV patients was severely reduced, approximately ten times less than controls. These observations were further confirmed by both diminished gene and protein expression of Foxp3 in the CD4(+)CD25(+) T cell population in PV patients. CONCLUSIONS: Numerical impairment of Treg cells may be involved in the pathogenesis of PV.

Possible role of VEGF in the progression of kidney disease in streptozotocin (STZ)-induced diabetic rats: effects of an ACE inhibitor and an angiotensin II receptor antagonist.

Two endothelium-derived factors, endothelin (ET), a vasoconstrictor, and vascular endothelial growth factor (VEGF), an angiogenic factor are thought to be involved in the pathogenesis of diabetic vascular complications. The aim of this study was to determine the effects of an angiotensin II type I (AT-1) receptor antagonist and an ACE inhibitor on the pathogenesis of VEGF and ET-1-mediated kidney disease in STZ-induced diabetic rats. Two days after STZ administration, diabetic rats were treated for 8 weeks with enalapril maleate, an ACE inhibitor, candesartan cilexetil, an AT-1 receptor antagonist, or saline. Urinary albumin and N-acetyl beta-D glucosaminidase (NAG) excretion as well as the VEGF protein content in the kidney were all found to be elevated in diabetic rats. Administration of enalapril maleate or candesartan cilexetil decreased the level of microalbuminuria and NAG excretion in diabetic rats. Administration of enalapril maleate also suppressed the elevated renal VEGF protein content in these animals while candesartan cilexetil treatment had no effect. Serum ET-1 and VEGF levels were unchanged by these treatments. These data support a role for AT-1 receptor antagonists and ACE inhibitors in the prevention of diabetic nephropathy, and suggest that the former may work by reducing renal VEGF levels.

A stable prostacyclin analogue reduces high serum TNF-alpha levels in diabetic patients.

AIMS: To confirm whether a prostacyclin (prostaglandin I (2)) affects the increased TNF-alpha concentration in sera of diabetic patients, we measured serum TNF-alpha concentration and treated these patients with oral administration of the stable prostacyclin analogue (Beraprost). Twelve of 20 type II diabetic patients were investigated for follow up-study and 6 of those patients were for therapy with Beraprost for diabetic neuropathy. SUBJECTS AND METHODS: Serum TNF-alpha concentration was quantified by EASIA using monoclonal antibodies directed against distinct epitopes of TNF-alpha. RESULTS: In diabetic patients, serum TNF-alpha concentration was significantly increased compared with that of healthy subjects. The augmented TNF-alpha concentration in these patients was not decreased by diabetic control using antihyperglycemic agents for 8 weeks but was reduced with oral administration of a stable prostacyclin (prostaglandin I (2)) analogue for 5 weeks without any changes of blood glucose levels. CONCLUSIONS: Stable prostacyclin analogue administration for a short term period reduced increased TNF-alpha levels in diabetic patients, not through the improved hyperglycemic condition but another pathway, probably a cAMP system. These results imply that treatment with the prostacyclin analogue may contribute to the prevention of progression in diabetic complications.

Changes of endothelin in streptozotocin-induced diabetic rats: effects of an angiotensin converting enzyme inhibitor, enalapril maleate.

Endothelin-1 (ET-1) concentrations are increased in patients with diabetes mellitus, particularly those with diabetic retinopathy, or essential hypertension. We hypothesized that ET-1 might participate in the development and progression of diabetic microangiopathy. In this study, the effects of the angiotensin converting enzyme (ACE) inhibitor, enalapril maleate, on diabetic angiopathy were examined in streptozotocin (STZ)-induced diabetic (STZ-DM) rats by monitoring variations in renal function and ET-1 concentrations in blood and organ tissues. Significant increases in kidney weight and in concentrations of urinary albumin, N-acetyl-fl-d-glucosamidase (NAG) and serum ET-1 were observed in the STZ-DM rats as compared with the non-diabetic rats, and the concentration of ET-1 in the kidneys tended to be increased. Microscopic and electron microscopic analyses showed increased mesangial cell proliferation, matrix expansion and enlarged mesangial area in the kidney of the diabetic rats. After administration of the ACE inhibitor, increased concentrations of urinary albumin and NAG in the STZ-DM rats were reduced to the control values with a slight improvement in the electron microscopic changes. These data suggest that ET-1 may be involved in the development and progression of diabetic nephropathy and may explain, in part, why diabetes is liable to complicate hypertension. ACE inhibitor may help to restore diabetic nephropathy in the STZ-induced diabetic rats.

Complete nucleotide sequence of the linear DNA plasmid pRS224 with hairpin loops from Rhizoctonia solani and its unique transcriptional form.

The complete nucleotide sequence of the linear DNA plasmid (pRS224-1) from the plant-pathogenic fungus Rhizoctonia solani isolate H-16 was determined; and its unique RNA transcripts were characterized. The pRS224-1 DNA consists of 4,986 nucleotides. A computer-based study of the folding of pRS224-1 at both termini predicted hairpin-loop structures. The hairpin loops consisted of the left and right termini of 236 and 264 nucleotides, respectively, and share no sequence homology. Unique poly(A) RNAs, 4.7 kb and 7.4 kb in length and hybridizing with the pRS224 DNA, were found in mycelial cells of R. solani H-16. Transcript product-mapping allowed the prediction of the locations of different expression signals. The 7.4-kb transcript is generated from the left terminal region of the complementary strand, via the full-length sense-strand, to the right terminal region of the complementary strand. The 4.7-kb transcript is generated from the center region of the sense strand to the right terminal region of the complementary strand. One open reading frame (ORF) found in pRS224-1 is 887 amino acids long and has a potential coding capacity of 102 kDa. The ORF contains the highly conserved domains characteristic of reverse transcriptase sequences, including the highly conserved YXDD sequence.

Effect of eicosapentaenoic acid ethyl ester on hypothyroid function.

Thyroid hormones affect reactions in almost all pathways of lipid metabolism. It has been reported that plasma free fatty acid (FFA) concentration in hypothyroidism is generally within the normal range. In this study, however, we show that plasma FFA concentration in some hypothyroid patients is higher than the normal range. Symptoms of thyroid dysfunction in these individuals were less severe than those of patients with lower plasma FFA concentrations. From these findings we hypothesized that the change in FFA concentration must correlate with thyroid function. Using an animal model, we then examined the effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E), a n-3 polyunsaturated fatty acid derived from fish oil, on thyroid function in 1-methyl-2-imidazolethiol (MMI)-induced hypothyroid rats. Oral administration of EPA-E inhibited reduction of thyroid hormone levels and the change of thyroid follicles in MMI-induced hypothyroid rats. These findings suggest that FFA may affect thyroid functions and EPA-E may prevent MMI-induced hypothyroidism.

The therapeutic effect of lipo PGE1 on diabetic neuropathy-changes in endothelin and various angiopathic factors.

A high blood concentration of endothelin (ET)-1 may participate in the onset and progress of diabetic microangiopathy, resulting in neuropathy. We examined the therapeutic effects of prostaglandin E1 (PGE1), which possesses both a peripheral vasodilating action and inhibition of platelet aggregation, on diabetic microangiopathy. Increases in both skin temperature and peripheral never conduction velocity in diabetic patients were recorded four weeks after Lipo PGE1 administration. A quantitative decrease in urinary albumin concentration was also observed, suggesting its efficacy of action was on diabetic nephropathy. Lipo PGE1 administration reduced the elevated circulating plasma ET-1 levels in the diabetic patients. As an increase in ET-1 concentrations is thought to correlate with the onset and progress of diabetic microangiopathy, the reduction of plasma ET-1 concentration by Lipo PGE1 administration may be one reason for the improvement in diabetic neuropathy and nephropathy.

Cryoglobulinemia in Japanese patients with chronic hepatitis C virus infection: host genetic and virological study.

Essential cryoglobulinemia is associated closely with hepatitis C virus (HCV) infection. The mechanism responsible for occurrence of the disease is unclear. The aim of this study was to investigate pathogenetic roles of HCV in cryoglobulinemia. One hundred sixty-seven consecutive patients with HCV were studied clinically by HCV grouping, HCV RNA levels, GBV-C/HGV, HCV quasispecies (target region was hypervariable region-1) and HLA polymorphism. The quasispecies in cryoprecipitate were compared with those in supernatant. The results of HLA polymorphism of patients with cryoglobulinemia were compared with those without cryoglobulinemia and healthy controls. The frequency of HCV-related cryoglobulinemia was 71 of 167 (42.5%). Patients with cirrhosis (36 of 63, 57.1%) had cryoglobulinemia more frequently than those with chronic hepatitis (35 of 104, 33.7%, P < 0.01). No significant differences were not found between the two groups (patients with and without cryoglobulinemia) in age, gender, HCV grouping, HCV RNA level and frequency of GBV-C/HGV. HCV was found quantitatively and clonally more frequently in the cryoprecipitate than in the supernatant. HLA polymorphism presented no significant differences among three groups. The stage of liver disease is one of pathogenetic factors. The greater the presence of HCV quasispecies in cryoprecipitate than in the supernatant indicates that various antigen presentations play an important role in the formation of cryoglobulin, whereas HLA typing dose not seem to contribute to the development of cryoglobulinemia.

Polymorphisms of the insulin gene among Japanese subjects.

We have sequenced the insulin gene in 72 unrelated Japanese subjects (52 with type 2 diabetes mellitus and 20 with normal glucose tolerance). We identified 6 mutations and all were found at a low frequency (1% to 4%). Three mutations were new. These included a C-to-G substitution in the promoter region, a G-to-A substitution in codon-2 resulting in an Ala-to-Thr replacement in amino acid -2 of the signal peptide, and a G-to-A substitution in intron 2. We have no evidence that any of the mutations that we found are the cause of diabetes. Thus, mutations in the insulin gene do not appear to be an important genetic factor contributing to the development of diabetes in this population.

Alteration of endothelin-1 concentration in STZ-induced diabetic rat nephropathy. Effects of a PGI(2) derivative.

BACKGROUND: Recently, an endothelin (ET-1) with a potent vasoconstrictive activity and stimulative activity of vascular muscular cell growth was discovered and blood ET-1 levels were higher in diabetic patients than in healthy subjects, suggesting that high ET-1 levels assist development and progression of diabetic microangiography. METHODS: We examined renal function, and serum and tissue ET-1 levels in streptozotocin (STZ)-induced diabetic rats treated with a prostaglandin (PG) I(2) derivative to investigate the effect of PGI(2) in diabetic vascular disturbance. RESULTS: Renal weight, urinary albumin, urinary N-acetyl-beta,D-glucosaminidase (NAG) and serum ET-1 levels increased in STZ-induced diabetic rats, and a tendency to increase in renal tissue ET-1 levels was observed. Furthermore, electron-microscopic findings in the kidneys showed mesangial cell proliferation and mesangial matrix expansion which might be caused by diabetic nephropathy. The PGI(2) derivative reduced urinary albumin and NAG levels in STZ-induced rats. It was considered, therefore, that the PGI(2) derivative is effective in diabetic nephropathy. As the PGI(2) derivative also reduced renal tissue ET-1 levels, improvement of diabetic nephropathy partially was considered to result from the reduction of renal tissue ET-1 levels. CONCLUSION: In STZ-induced rats, increased serum ET-1 levels and a tendency to increase in renal tissue ET-1 levels were associated with increases in urinary albumin and NAG levels, and these levels were decreased by a PGI(2) derivative. These findings suggested that increased ET-1 concentrations assist development and progression of diabetic nephropathy, especially diabetic microangiopathy, and the PGI(2) derivative may be effective for inhibition of diabetic microangiopathy mediated by reduction of ET-1 concentrations.

Clinical usefulness of intravenous albunex for the Doppler assessment of aortic stenosis.

Optimal Doppler recordings of stenotic aortic flow are not always easy to obtain. Therefore, the present study investigated how useful intravenous Albunex injections were for improving the Doppler assessment of pressure gradients for aortic stenosis in 20 consecutive patients who underwent Doppler and left-heart catheterization studies within a 1-week period. Continuous-wave Doppler echocardiography was performed using both a 2.5 MHz duplex and a 1.9MHz independent transducer before and after Albunex injections. The maximum and mean pressure gradients were calculated from the highest Doppler velocity tracings using the simplified Bernoulli equation. Pullback catheterization pressure tracings from the left ventricle to the ascending aorta were superimposed for determination of the maximum instantaneous and mean pressure gradients. The Doppler-derived peak and mean pressure gradients showed significant underestimation compared with the catheterization gradients (23+/-17 mmHg and 11+/-7 mmHg, respectively). However, this underestimation disappeared with Albunex injection (-2+/-7 mmHg and -1+/-4mmHg, respectively). Although the Doppler-derived instantaneous and mean pressure gradients correlated well with the catheterization gradients (r=0.909 and r=0.879, respectively), they became much closer with Albunex (r=0.987 and r=0.963, respectively). The improvements in the Doppler-derived peak pressure gradients were significant from an apical window (n=12, 84-120mmHg, p<0.001). but less so from non-apical windows (n=8, 84-91 mmHg, p=0.0146). Accordingly, Albunex is most useful for Doppler recordings of stenotic aortic flow available from the apical window, but not less so from other acoustic windows.

Reverse transcriptase is elevated in the thyroid tissue from Graves' disease patients.

OBJECTIVE: Hypertrophy of the thyroid gland in Graves' disease is related to an autoimmune response directed against TSH receptors found in thyroid cells. Recently, investigators have suggested that autoimmune diseases, including thyroid diseases may, at least in part, correlate with the expression of proteins encoded by the retroviral genome. In the present study, to confirm the correlation between thyroid autoimmune disorders and retroviral infections, we examined reverse transcriptase (RT) activity in thyroid tissues as a marker of retroviral infection. PATIENTS AND MEASUREMENTS: Thyroid tissues obtained at surgery from patients with various thyroid disorders (normal thyroid adjacent to adenoma, six cases; Graves' disease thyroid tissue, 25 cases; adenoma, eight cases; papillary carcinoma, 12 cases; Graves' disease peripheral blood lymphocytes, 11 cases) were used for RT assay, using a specific, improved assay system. RESULTS: Thyroid tissue extracts from patients with Graves' disease contained high RT activity which resembled that demonstrated in retroviruses. The RT existed in the thyroid tissue as a complex, with endogenous template RNA, and the activity was confirmed not to be due to other DNA polymerases. CONCLUSION: Retroviral RT distinguished from known cellular DNA polymerases is expressed in the thyroids of patients with Graves' disease. In a permissive genetice and immunological environment, retroviral DNA integrated into genomic DNA could precipitate the onset of Graves' disease.

Production of IL-10 and IL-12 in CD40 and interleukin 4-activated mononuclear cells from patients with Graves' disease.

We investigated the effect of T cell-dependent B cell activation on the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMCs) obtained from patients with Graves' disease vs Hashimoto's thyroiditis, type 1 diabetes or normal controls. Incubation of PBMCs, from each of the subject groups, with a combination of anti-CD40 monoclonal antibodies and interleukin 4 (IL-4)-activated B cells, as shown by an increased level of soluble CD23. There was also a notable increase in the number of CD23(+)cells in PBMCs from patients with Graves' disease as compared to the other subject groups. This combination of B cell stimulants increased production of IL-10 in PBMCs obtained from patients with Graves' disease relative to those patients with Hashimoto's thyroiditis, type 1 diabetes, or the control subjects. The production of IL-12 showed wide variation that depended on the basal IL-12 level. In subjects with a low basal IL-12 level there was a positive correlation between the production of IL-12 and that of IL-10 from PBMCs stimulated with anti-CD40 antibodies plus IL-4. On the contrary, in the patients with a high basal IL-12 level, no change or a decrease of IL-12 production was observed after the stimulation. Thus, T cell-dependent B cell activation via a CD40 pathway triggers the overproduction of IL-10 and overcome the effect of IL-12 to shift the Th(1)/Th(2)balance to Th(2)dominance in patients with Graves' disease but not in Hashimoto's thyroiditis or type 1 diabetes.

Scavenging effect of nicorandil on free radicals and lipid peroxide in streptozotocin-induced diabetic rats.

Free radicals and lipid peroxide (LPO), easily formed in the diabetic state, play an important role in the development of diabetic complications. Potentially, nicorandil may reduce the production of free radicals and LPO in various organs. In fact, increased LPO levels in the serum, kidney, and cardiac muscle of diabetic (DM) rats were reduced by nicorandil treatment (N treatment). Xanthine oxidase (XOD), which produces free radicals, was decreased in the liver and increased in the kidney of DM rats compared with control rats, and these changes were prevented by N treatment. The concentration of Cu, Zn-superoxide dismutase (SOD) decreased in the cardiac muscle and increased in the kidney of DM rats, and these changes returned to normal after N treatment. The decreased concentration of Mn-SOD in the liver, kidney, and cardiac muscle from DM rats was also reversed by N treatment. The changes in catalase and glutathione peroxidase (GSH-PX) activities in DM rats were not improved effectively by N treatment. Another K-adenosine triphosphate (K-ATP) channel opener, tilisolol hydrochloride, had an effect similar to that of nicorandil. The effects of nicorandil and tilisolol were studied only in DM rats. These data imply that N treatment, as an antioxidative therapy, may be beneficial in preventing diabetic complications due to lipoperoxidation and free radicals in DM rats.

Lipid peroxidation levels in rat cardiac muscle are affected by age and thyroid status.

Free radicals, hydroxyperoxides and H(2)O(2) are all known to damage cell components. This study was designed to compare the concentrations of hydroxyperoxide and free radical scavengers in the cardiac muscles of old rats in the hyper- or hypothyroid condition, to determine whether rates of peroxidation would differ with age, thyroid status, or both. Rats were rendered hyper- or hypothyroid by administration of l-thyroxine or methimazole for 4 weeks. Among the old rats, the lipid peroxide (LPO) concentrations, measured as thiobarbituric acid (TBA) reactants, were significantly greater in the hyperthyroid than in the euthyroid state and the LPO concentrations measured as TBA+Fe(3+) reactants, which may be precursors of LPO, were significantly greater in the hyperthyroid state, whereas in young rats, the LPO concentrations measured by TBA or TBA+Fe(3+) methods did not differ significantly in the hyperthyroid state. In the euthyroid state, the concentration of LPO measured as TBA+Fe(3+) reactants was significantly reduced with age. Xanthine oxidase (XOD) activity also was markedly increased with age, being more pronounced in the hyperthyroid than in the euthyroid state. The Mn and Cu/Zn superoxide dismutase activities were greater in the hyperthyroid than in the euthyroid state. Glutathione peroxidase activity decreased with age in the euthyroid and, particularly, in the hyperthyroid state. Catalase activity was not affected in the old rats. Concentrations of alpha-tocopherol in the old rats were high in the hyperthyroid state and low in the hypothyroid state, whereas the levels of beta- and gamma-tocopherols in these rats were unchanged in both conditions as compared with the euthyroid state findings. Data suggest that the site of free radical generation differs in older rats, with additional shifts in the location of intracellular lipid peroxidation being noted during hyperthyroidism. Thus, as rats age, the reduction of the free radical scavenger system and the increase in LPO and XOD activities might induce myocardial dysfunction.