Occlusal and TMJ loads in subjects with experimentally shortened dental arches.

To determine whether shortened dental arches (SDAs) cause functional overloading of the teeth and the temporomandibular joints, which has been implicated in periodontal diseases and temporomandibular disorders, we investigated the influences of SDA on occlusal and joint loads. Bite force and masticatory muscle electromyograms were recorded in five dentate subjects who clenched maximally on intra-oral appliances, creating symmetrical SDAs experimentally. Muscular forces estimated from the recorded electromyograms were fed into a finite element jaw model for calculating bite forces and joint loads. Comparison between the measured and the calculated bite forces ensured that the joint loads were representative. The bite force on each tooth increased with missing molar occlusions, while joint loads decreased. The bite force per root surface area was always greatest on the most posterior tooth, and these values were most constant. The findings provide no evidence that SDA causes overloading of the joints and the teeth, which suggests that neuromuscular regulatory systems are controlling maximum clenching strength under various occlusal conditions.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1beta (IL-1beta) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 x g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1beta, and this may contribute to inflammatory reactions in the CNS.

Hammerhead ribozyme targeting human platelet-derived growth factor A-chain mRNA inhibited the proliferation of human vascular smooth muscle cells.

Platelet-derived growth factor (PDGF) A-chain contributes to the pathogenesis of cardiovascular proliferative diseases, such as hypertensive vascular disease, atherosclerosis, and re-stenosis of an artery after angioplasty. To develop a ribozyme against human PDGF A-chain mRNA as a gene therapy for human arterial proliferative diseases, we designed and synthesized a 38-base hammerhead ribozyme to cleave human PDGF A-chain mRNA at the GUC sequence at nucleotide 591. In the presence of MgCl(2), synthetic hammerhead ribozyme to human PDGF A-chain mRNA cleaved the synthetic target RNA to two RNA fragments at a predicted size. Doses of 0.01-1.0 microM hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited angiotensin II (Ang II) and transforming growth factor (TGF)-beta(1)-induced DNA synthesis in vascular smooth muscle cells (VSMC) from human in a dose-dependent manner. One micromolor of hammerhead ribozyme to human PDGF A-chain mRNA significantly inhibited Ang II-induced PDGF A-chain mRNA and PDGF-AA protein expressions in VSMC from humans. These results indicate that the designed hammerhead ribozyme to human PDGF A-chain mRNA effectively inhibited growth of human VSMC by cleaving the PDGF A-chain mRNA and inhibiting the PDGF-AA protein expression in human VSMC. This suggests that the designed hammerhead ribozyme to PDGF A-chain mRNA is a feasible gene therapy for treating arterial proliferative diseases.

Endogenous angiotensin II suppresses insulin signaling in vascular smooth muscle cells from spontaneously hypertensive rats.

BACKGROUND: Angiotensin II (Ang II) has been reported to inhibit insulin signaling at multiple levels in vascular smooth muscle cells (VSMC) in vitro. We have demonstrated that VSMC from spontaneously hypertensive rats (SHR) produce Ang II in a homogeneous culture. OBJECTIVE: In the current study, we investigated influences of endogenous Ang II on insulin signaling in VSMC from SHR. DESIGN AND METHODS: Phosphatidylinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were measured in VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats in the absence and presence of Ang II type 1 receptor antagonist RNH6270 and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor U0126. RESULTS: Insulin treatment increased PI3-kinase activity in VSMC from WKY rats in a dose-dependent manner. In contrast, insulin treatment of VSMC from SHR did not affect PI3-kinase activity. However, co-treatment of VSMC from SHR with RNH6270 and insulin, increased PI3-kinase activity. PI3-kinase activity, IRS-1-associated tyrosine phosphorylation and p85 subunit of PI3-kinase in VSMC from WKY rats decreased in response to treatment with Ang II and returned to control levels upon co-treatment with U0126. Basal levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase were significantly lower in VSMC from SHR than in cells from WKY rats. U0126 treatment of VSMC from SHR significantly increased levels of PI3-kinase activity, IRS-1-associated tyrosine phosphorylation, and p85 subunit of PI3-kinase. CONCLUSION: These results indicate that endogenous Ang II suppresses insulin signaling in VSMC from SHR by activating extracellular signal-regulated kinase. These findings suggest that tissue Ang II may play a role in insulin resistance in hypertension.

Role of endogenous angiotensin II in the increased expression of growth factors in vascular smooth muscle cells from spontaneously hypertensive rats.

In culture, vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) show exaggerated growth compared with cells from normotensive Wistar-Kyoto (WKY) rats. SHR-derived VSMC express higher levels of transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF) A-chain, and basic fibroblast growth factor (bFGF) mRNAs than cells from WKY rats. We have recently observed production of angiotensin II (Ang II) in homogeneous cultures of VSMC from SHR. In the current study we investigated the contribution of endogenous Ang II to increased expression of the above-mentioned growth factors in VSMC from SHR. The levels of mRNAs encoding TGF-beta1, PDGF A-chain, and bFGF were determined by reverse transcription-polymerase chain reaction and were much higher in VSMC from SHR than in cells from WKY rats. The basal level of Ang II-like immunoreactivity (LI) in conditioned medium as determined by radioimmunoassay was significantly higher in VSMC from SHR than in cells from WKY rats. Isoproterenol is known to induce angiotensinogen gene significantly increased Ang II-LI in VSMC from both WKY rats and SHR. Isoproterenol also increased angiotensinogen, TGF-beta1, PDGF A-chain, and bFGF mRNAs in VSMC from SHR. An angiotensin-converting enzyme inhibitor delapril significantly decreased Ang II-LI in VSMC from WKY rats and SHR. Delapril considerably decreased the levels of TGF-beta1, PDGF A-chain, and bFGF mRNAs in VSMC from SHR. An Ang II type 1 receptor antagonist CV 11974 decreased the levels of TGF-beta1, PDGF A-chain, and bFGF mRNAs, and the levels of TGF-beta1, PDGF-AA, and bFGF proteins in VSMC from SHR. These findings suggest that increased generation of Ang II is associated with enhanced expression of TGF-beta1, PDGF A-chain, and bFGF, and the increases in the levels of these growth factors by endogenous Ang II may contribute to the exaggerated growth of VSMC from SHR.

Identification of base substitutions in ten types of rare variants of phosphoglucomutase-1 (PGM1) encountered in Japanese.

In a previous starch-gel electrophoresis study of erythrocyte phosphoglucomutase-1 (PGM1) in 23,095 Japanese from Hiroshima and Nagasaki, we detected 14 types of rare variant alleles. To determine sequence differences in these rare alleles, cell lines were established from peripheral B-lymphocytes from 24 unrelated individuals in whom nine types of rare variants are presumed to exist on the basis of earlier electrophoresis studies. cDNAs reverse transcribed from mRNAs extracted from these cell lines were amplified by polymerase chain reaction and sequences determined. Amino acid substitution types were deduced from each cDNA sequence. Although two individuals were reported to have an identical electromorph (PGM1 4HR3), sequence analysis revealed that alleles encoding these electromorphs possessed different base substitutions, and one was renamed PGM1 4HR4. As the amino acid substitution of ten different variants could be deduced by cDNA sequence in this study, the effect of each amino acid substitution on enzyme activity could be precisely simulated. The secondary structure of each variant predicted by computer simulations revealed that very decreased activity observed on PGM1 4HR2 protein was caused by significant secondary structure change introduced by the amino acid substitution. On the basis of the crystal structure, the amino acid substitutions of the ten types of rare variants seem to be outside the active center of this enzyme.

Angiotensin II regulates the cell cycle of vascular smooth muscle cells from SHR.

We have demonstrated that spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show the exaggerated growth and produce angiotensin II (Ang II). In the current study, we investigated the role of endogenous Ang II in the regulation of the cell cycle in VSMC from SHR. Levels of Ang II in conditioned medium from SHR-derived VSMC cultured without serum were significantly higher than levels in conditioned medium from Wistar-Kyoto (WKY) rat-derived VSMC. Basal DNA synthesis was higher in quiescent VSMC from SHR than that in cells from WKY rats. An Ang II type 1 receptor antagonist, CV11974, significantly inhibited the elevation in DNA synthesis in quiescent VSMC from SHR but did not affect it in cells from WKY rats. Cellular DNA content analysis by flow cytometry revealed that the proportion of cells in S phase was higher, whereas the proportion of cells in G1+G0 phase was lower in VSMC from SHR than those in cells from WKY rats. CV11974 significantly decreased the proportion of cells in S phase and correspondingly increased the proportion of cells in G1+G0 phase in VSMC from SHR, but it did not affect the proportion in cells from WKY rats. Cyclin-dependent kinase 2 (CDK2) activity, which is known to induce the progression from G1 to S phase, was higher in VSMC from SHR than in cells from WKY rats. Expression of CDK2 inhibitor p27(kip1) mRNA was markedly higher in VSMC from SHR than in cells from WKY rats. CV11974 decreased expression of p27(kip1) mRNA in VSMC from SHR, whereas CV11974 increased it in cells from WKY rats. These findings indicate that enhanced production of endogenous Ang II regulates the cell cycle especially in the progression from G1 to S phase, and increases CDK2 activity, which is independent of p27(kip1) in VSMC from SHR.

Phenotypic modulation by fibronectin enhances the angiotensin II-generating system in cultured vascular smooth muscle cells.

We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II-generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0. 2% calf serum for 48 hours. Immunofluorescence and protein levels of alpha-smooth muscle (SM) actin and expression of SM22alpha mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 microg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II-like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II-generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.

Effect of methylene methylimino linkage of antisense oligonucleotide to the platelet-derived growth factor A-chain on growth of vascular smooth muscle cells from spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells show exaggerated growth and increased expression of platelet-derived growth factor (PDGF) A-chain mRNA. We examined the effect of methylene methylimino linkage of antisense oligodeoxynucleotide, a novel modification of antisense oligodeoxynucleotide designed to increase nuclease resistance, to PDGF A-chain on the exaggerated growth of vascular smooth muscle cells from SHR. Methylene methylimino-linked oligodeoxynucleotide provided complete resistance against S1 nuclease. Methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain resulted in a rapid inhibition of basal DNA synthesis of vascular smooth muscle cells from SHR. This inhibition was much greater than that produced by phosphorothioate linkage of antisense oligodeoxynucleotide to PDGF A-chain. The methylene methylimino linkage of antisense oligodeoxynucleotide to PDGF A-chain may prove useful in the treatment of arterial proliferative diseases including hypertension.

Angiotensin II upregulates transforming growth factor-beta type I receptor on rat vascular smooth muscle cells.

Angiotensin II (Ang II) and transforming growth factor-beta (TGF-beta) modulate cell growth and metabolism. Our objective was to evaluate the effect of Ang II on the characteristics and expression of TGF-beta receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto rats. The addition of TGF-beta1 elicited a biphasic response on DNA synthesis in cultured VSMC in the absence of Ang II, but TGF-beta1 did not stimulate DNA synthesis in the presence of Ang II. TGF-beta binding data showed that Ang II increased the specific binding of 125I-TGF-beta1 by enhancing the expression of lower affinity receptors and increasing the number of binding sites. Ang II alone did not stimulate DNA synthesis in these cultures. However, Ang II significantly stimulated DNA synthesis after the inhibition of endogenous TGF-beta with a neutralizing antibody. The DNA synthesis stimulated by phorbol ester milisterol (PMA) was not affected by the TGF-beta neutralizing antibody. Affinity labeling data revealed receptor-ligand complexes of 280, 85, and 70 kDa, corresponding to TGF-beta type III, II, and I receptors, respectively. Incubation of VSMC with Ang II but not with PMA markedly increased the expression of the TGF-beta type I receptor. Reverse transcription and polymerase chain reaction data also indicated that Ang II, but not PMA, significantly increased the expression of TGF-beta type I receptor mRNA. Results suggest that Ang II increases the binding of TGF-beta with upregulation of TGF-beta type I receptor via a C-kinase-independent pathway. The enhanced expression of the TGF-beta type I receptor may counteract Ang II-promoted growth of VSMC.

Low dose of eicosapentaenoic acid inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats through suppression of transforming growth factor-beta.

OBJECTIVE: To evaluate effects of eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, on the exaggerated growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). DESIGN: Cultured VSMC were prepared by an explant method from thoracic aortas in 8-week-old male Wistar-Kyoto (WKY)/Izumo rats and SHR/Izumo. Effects of EPA on basal DNA synthesis, expression of growth factors and cyclin-dependent kinase 2 (cdk2) activity were examined in VSMC from WKY rats and SHR. METHODS: The cell cycles were synchronized with serum deprivation, then DNA synthesis in VSMC was measured by [3H]-thymidine incorporation. Fatty acid composition of the phospholipid fraction in VSMC was measured by gas chromatography. Expression of platelet-derived growth factor (PDGF) A-chain, transforming growth factor (TGF)-beta1 and basic fibroblast growth factor (bFGF) mRNAs was evaluated by reverse-transcription and polymerase chain reaction analysis. Cdk2 activity was determined by autoradiography after polyacrylamide gel electrophoresis of VSMC extracts that had been immunoprecipitated with anti-cdk2 antibody and protein A sepharose, and then incubated with 32P-ATP and histone H1. RESULTS: High concentrations (40 and 80 micromol/I) of EPA significantly inhibited basal DNA synthesis in VSMC from both rat strains. Low dose (20 micromol/l) of EPA significantly inhibited basal DNA synthesis in VSMC from SHR, whereas the same dose of EPA stimulated DNA synthesis in VSMC from WKY rats. In analysis of fatty acid composition, low dose of EPA was considerably incorporated in VSMC. Low dose of EPA significantly inhibited angiotensin II- and phorbol ester milisterol-stimulated DNA synthesis in VSMC from both rat strains, whereas EPA did not affect PDGF-AA-stimulated DNA synthesis in VSMC from either rat strain. Low dose of other polyunsaturated fatty acids such as docosahexaenoic acid, arachidonic acid and linoleic acid did not significantly affect basal DNA synthesis in VSMC from either strain. Low dose of EPA significantly inhibited expression of TGF-beta1 mRNA in VSMC from SHR, whereas EPA did not affect expression of PDGF A-chain and bFGF mRNAs in VSMC from SHR. Cdk2 activity in VSMC from SHR was higher than that from WKY rats. Low dose of EPA inhibited cdk2 activity in VSMC from SHR, whereas it stimulated the activity in VSMC from WKY rats. CONCLUSION: Low dose of EPA exerted specific inhibition of the exaggerated growth of VSMC from SHR through the suppression of TGF-beta.

Chimerism in humans after intragenic recombination at the haptoglobin locus during early embryogenesis.

The human haptoglobin (HP) HP*2 allele contains a 1.7-kilobase (kb) intragenic duplication that arose after a unique nonhomologous recombination between the prototype HP*1 alleles. During a genetic screening of 13,000 children of survivors exposed to atomic-bomb radiation and 10,000 children of unexposed persons, two children suspected of carrying de novo mutations at the haptoglobin locus were identified (one in each group). DNA analyses of single-cell-derived colonies of Epstein-Barr virus-transformed B cells revealed that the two children were mosaics comprising HP*2/HP*2 and HP*2/HP*1 cells at a ratio of approximately 3:1. We infer that the latter cells are caused by reversion of one HP*2 allele to HP*1 through an intramolecular homologous recombination between the duplicated segments of the Hp*2 allele that excised one of the segments. Because the mosaicism is substantial (approximately 25%), this recombination must have occurred in early embryogenesis. The frequency of finding these children and the extent of their mosaicisms corresponds to an HP*2 to HP*1 reversion rate of 8 x 10(-6) per cell during development. This leads to the prediction that the HP*1 allele also will be represented, although usually at a very low frequency, in any HP2-2 person. We tested this prediction by using PCR for a single individual and found the HP*1 allele at frequencies of 4 x 10(-6) and 3 x 10(-6) in somatic and sperm cells. The HP*1 allele was detected by PCR in all four other HP2-2 individuals, which supports the regular but rare occurrence somatically of homologous recombination within duplicated regions in humans, in agreement with previous observations in mouse and Drosophila.

Contribution of synthetic phenotype on the enhanced angiotensin II-generating system in vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVE: We have demonstrated that cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR), but not from normotensive Wistar-Kyoto (WKY) rats, produce angiotensin II (Ang II) in a homogeneous culture with increased levels of angiotensinogen, cathepsin D and angiotensin converting enzyme (ACE) at early passages. In the current study, we investigated how changes in the cell phenotype affect the Ang II-generating system and the growth of VSMC from SHR. DESIGN AND METHODS: We evaluated basal DNA synthesis by [3H]thymidine incorporation, immunofluorescence of alpha-smooth muscle (SM) actin, mRNA expression of phenotype markers such as SM22alpha appeared by contractile phenotype, Ang II-generating system components and growth factors by reverse transcription and polymerase chain reaction analysis, and Ang II levels by radioimmunoassay in quiescent VSMC from WKY/Izumo rats and SHR/Izumo at passages 4, 8 and 12. RESULTS: Basal DNA synthesis in VSMC from WKY rats increased with increasing passage number, whereas in cells from SHR it was markedly higher at early passages and was not affected by the passages. At early passage numbers, immunofluorescence of alpha-SM actin was stronger in VSMC from WKY rats than in cells from SHR, but decreased after several passages. Expression of SM22alpha mRNA was higher in VSMC from WKY rats than in cells from SHR at early passages, and decreased after several passages in cells from both rat strains. Expression of matrix Gla mRNA was higher in VSMC from SHR than in cells from WKY rats at early passage, and increased after several passages in cells from both rat strains. Ang II was not detected at early passages but increased in VSMC from WKY rats with increasing passage, whereas it was detected in VSMC from SHR at early passages and did not change with the passages. Expression of angiotensinogen mRNA was higher in VSMC from SHR than in cells from WKY rats, and was not affected by the passages. Expressions of cathepsin D and ACE mRNA were higher in VSMC from SHR than in cells from WKY rats at early passage, and were increased by the passages in VSMC from WKY rats. Expressions of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNA were significantly higher in VSMC from SHR than in cells from WKY rats, and were increased by the passages. CONCLUSION: These data indicate that early in culture VSMC from SHR have the synthetic phenotype, whereas VSMC from WKY rats have the contractile phenotype which then changes to the synthetic phenotype after increased passage numbers, with increased expression of cathepsin D and ACE, which produce Ang II, and increased expression of Ang II-related growth factors, which induce the exaggerated growth observed in VSMC from SHR.

Elevated plasma immunoreactive neuropeptide Y concentrations and its increased urinary excretion in patients with advanced diabetic nephropathy.

Neuropeptide Y (NPY) is a potent vasoconstrictor peptide that is abundant in the brain and the peripheral sympathetic nervous system. In the present study we investigated possible changes in plasma immunoreactive (IR)-NPY concentrations and urinary IR-NPY excretion in patients with non-insulin dependent diabetes mellitus (NIDDM) and the relationship to diabetic complications, such as nephropathy and neuropathy. IR-NPY in plasma and urine was measured by radioimmunoassay in 69 patients with NIDDM. Plasma IR-NPY concentrations in patients with advanced nephropathy (creatinine clearance <30 ml/min) (100.5 +/- 10.3 pmol/l, n=9, mean +/- SEM) were higher than in the control subjects (55.0 +/- 6.8 pmol/l, n=15) (P<0.02). Urinary excretion of IR-NPY and fractional excretion of NPY were also increased in the patients with advanced nephropathy. Sephadex G-50 column chromatography of the urine extracts obtained from healthy subjects, diabetic patients with renal failure and non-diabetic patients with renal failure showed an immunoreactive peak eluting in the NPY position. On the other hand, neither plasma nor urinary IR-NPY was high in patients with retinopathy, or in patients with peripheral neuropathy. The present study has, for the first time, shown high plasma IR-NPY concentrations and urinary IR-NPY excretion in NIDDM patients with advanced nephropathy.

Production of angiotensin II by homogeneous cultures of vascular smooth muscle cells from spontaneously hypertensive rats.

Production of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) has now been investigated. A nonpeptide antagonist (CV-11974) of Ang II type 1 receptors inhibited basal DNA synthesis in VSMC from SHR, but it had no effect on cells from Wistar-Kyoto (WKY) rats. Ang II-like immunoreactivity, determined by radioimmunoassay after HPLC, was readily detected in conditioned medium and extracts of SHR-derived VSMC, whereas it was virtually undetectable in VSMC from WKY rats. Isoproterenol increased the amount of Ang II-like immunoreactivity in conditioned medium and extracts of SHR-derived VSMC, whereas the angiotensin-converting enzyme inhibitor delapril significantly reduced the amount of Ang II-like immunoreactivity in conditioned medium and extracts of these cells. Reverse transcription-polymerase chain reaction analysis revealed that the abundance of mRNAs encoding angiotensinogen, cathepsin D, and angiotensin-converting enzyme was greater in VSMC from SHR than in cells from WKY rats. The abundance of cathepsin D protein by Western blotting was greater in VSMC from SHR than in cells from WKY rats. Ang I-generating and acid protease activities were detected in VSMC from SHR, but not in cells from WKY rats. These results suggest that SHR-derived VSMC generate Ang II with increases in angiotensinogen, cathepsin D, and angiotensin-converting enzyme, which contribute to the basal growth. Production of Ang II by homogeneous cultures of VSMC is considered as a new mechanism of hypertensive vascular disease.

High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli.

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.

Incidence and distribution pattern of pelvic and paraaortic lymph node metastasis in patients with Stages IB, IIA, and IIB cervical carcinoma treated with radical hysterectomy.

BACKGROUND: The incidence and distribution pattern of retroperitoneal lymph node metastasis in patients with cervical carcinoma should be investigated based on data from systematic pelvic lymph node (PLN) and paraaortic lymph node (PAN) dissection, so that a basis can be established for determining the site of selective lymph node dissection or sampling. METHODS: A total of 208 patients with Stages IB, IIA, and IIB cervical carcinoma who underwent radical hysterectomy and systematic pelvic and PAN dissection were investigated for lymph node metastasis and histopathologic risk factors for lymph node metastasis. RESULTS: Fifty-three patients (25.5%) had lymph node metastasis. The obturator lymph nodes were most frequently involved, with a rate of 18.8% (39/208). Forty-nine of 53 node-positive patients had lymph node metastasis in the obturator, internal iliac, or common iliac lymph nodes. Of 26 solitary lymph node metastases confined to one node group, 18 were in the obturator, 3 in the internal iliac, 3 in the parametrial, and 2 in the common iliac lymph nodes. A multiple logistic regression analysis revealed that deep cervical stromal invasion and lymph-vascular space invasion were related to PLN metastasis. It was also shown that metastasis to bilateral PLNs (excluding the common iliac lymph nodes) as well as metastasis to the common iliac lymph nodes were significantly related to PAN metastasis. CONCLUSIONS: The results of this study suggest that the obturator lymph nodes can be sentinel lymph nodes of cervical carcinoma. PAN metastasis appears to occur secondarily to wide-spread PLN metastasis. These results provide a basis for determining the site of selective lymph node dissection and for estimating the existence of PAN metastasis from the pattern of metastasis in PLN in patients with cervical carcinoma.

Distribution pattern and risk factors of pelvic and para-aortic lymph node metastasis in epithelial ovarian carcinoma.

The distribution of lymph node metastasis and the clinicopathologic risk factors for nodal involvement in ovarian carcinoma need to be clarified based on systematic lymph node dissection. We studied 115 patients with ovarian carcinoma who underwent systematic pelvic and para-aortic lymph node dissection between 1987 and 1997. The incidence and distribution of lymph node metastasis are described and the clinico-pathologic risk factors for nodal involvement are investigated. Based on the occurrence of lymph node metastasis in the early stages, the incidence of solitary node involvement and the distribution of lymph node metastasis, we conclude that the primary site of nodal involvement in ovarian carcinoma is the para-aortic node (PAN), especially PAN superior to the inferior mesenteric artery (IMA). By univariate analysis, clinical stage, histologic type (mucinous vs. others), grade, multiple peritoneal metastases, peritoneal cytology, volume of ascites and serum CA125 level were correlated with overall incidence of lymph node metastasis. By performing a multivariate analysis with the clinical stage excluded, it was revealed that grade and peritoneal cytology were independent factors for PAN metastasis (p < 0.0025 and < 0.001, respectively) and that multiple peritoneal metastases and PAN metastasis were significant predictors of pelvic node metastasis (p < 0.01 and < 0.005, respectively). In conclusion, the PANs superior and inferior to IMA should be explored in staging of ovarian carcinoma that appears to be confined to the ovaries. To determine accurately the extent of disease, both the para-aortic and pelvic areas may need to be sampled or dissected in the case of ovarian carcinoma involving the peritoneal surfaces.