Biomedical subjects

H Nagase

Publications and source records attributed to H Nagase.

At least 181 records · Page 10Linked to original sources

PubMed1998

Inhibition effect of propargylglycine on human fibrosarcoma HT-1080 cell invasiveness.

Effect of propargylglycine (2-Amino-4-pentynoic acid, PPG) on invasive property of human fibrosarcoma HT-1080 cell was investigated. PPG treatment of HT-1080 significantly reduced the total cellular metallothioneins (MTs) contents, and the resistance of HT-1080 against heavy metals toxicity decreased with the decrease of the MTs contents. The HT-1080 cell invasion to reconstituted basement membrane Matrigel (MG) was inhibited by the PPG treatment in a PPG concentration-dependent fashion. The inhibition was due to the lowering of HT-1080 cells attachment to MG and degradation activity of matrix metalloproteinases (MMPs) secreted from HT-1080 by the PPG treatment. However, the chemotactic ability of the PPG treated HT-1080 was enhanced. Our results suggest that MTs concentration levels in a malignant tumor cell are closely related to its invasiveness, and if MTs level of tumor cell can be controlled, cancer metastasis may be able to be controlled.

Alkynes↗

PubMed1997

Antinociceptive effects of (+)-matrine in mice.

The antinociceptive potency of matridin-15-one ((+)-matrine) was examined using the acetic acid-induced abdominal contraction test and the tail-flick test in mice. (+)-Matrine, at doses of 1 to 10 mg/kg, s.c., produced a marked and dose-dependent inhibition of the number of acetic acid-induced abdominal contractions in mice. The antinociceptive effect of (+)-matrine in the acetic acid-induced abdominal contraction test in mice was identical to that of pentazocine. Indeed, there was no significant difference in the ED50 (mg/kg with 95% confidence limits) values for the inhibition of acetic acid-induced abdominal contractions between (+)-matrine (4.7 (4.1-5.3)) and pentazocine (3.3 (2.2-5.0)). Furthermore, in the tail-flick assay, (+)-matrine at doses of 10 and 30 mg/kg, s.c., again produced a dose-dependent antinociceptive effect. When nor-binaltorphimine (20 mg/kg, s.c.), a selective kappa-opioid receptor antagonist, was administered 3 h before treatment with (+)-matrine, the antinociceptive effect of (+)-matrine was markedly antagonized. Furthermore, the antinociceptive effect of (+)-matrine was partially antagonized by pretreatment with beta-funaltrexamine, a selective mu-opioid receptor antagonist. Naltrindole, a selective delta-opioid receptor antagonist, had no effect on the antinociceptive effect of (+)-matrine. In conclusion, (+)-matrine produced an antinociceptive effect mainly through the activation of kappa-opioid receptors and partially through mu-opioid receptors.

Abdomen↗

PubMed1997

Modification of morphine-induced place preference by diabetes.

The effects of diabetes on morphine-induced place preference in mice were examined. Morphine caused dose-related place preference in both diabetic and non-diabetic mice. This morphine-induced place preference in diabetic mice was greater than that in non-diabetic mice. The morphine (5 mg/kg)-induced place preference in both diabetic and non-diabetic mice was significantly antagonized by pretreatment with beta-funaltrexamine, a selective mu-opioid receptor antagonist, but not with naloxonazine, a selective mu1-opioid receptor antagonist. The morphine (5 mg/kg)-induced place preference in non-diabetic mice was attenuated by pretreatment with either naltriben, a selective delta2-opioid receptor antagonist, or 7-benzylidenenaltrexone. a selective delta1-opioid receptor antagonist. Moreover, the morphine (10 mg/kg)-induced place preference in non-diabetic mice was antagonized by pretreatment with 7-benzylidenenaltrexone (0.7 mg/kg). Although 7-benzylidenenaltrexone had no effect on the place preference induced by 5 mg/kg morphine in diabetic mice, it reduced the place preference induced by 3 mg/kg morphine. Furthermore, the morphine (5 mg/kg)-induced place preference in diabetic mice was significantly antagonized by co-pretreatment with beta-funaltrexamine (10 mg/kg) and 7-benzylidenenaltrexone (0.7 mg/kg). 2-Methyl-4a alpha-(3-hydroxyphenyl)- 1,2,3,4,4a,5,12,12a alpha-octahydroquinolino[2,3,3-g]isoquinoline (TAN-67), a non-peptide delta-opioid receptor agonist, produced place preference in diabetic, but not in non-diabetic mice. These results support the hypothesis that the morphine-induced place preference is mainly mediated through the activation of the mu2-opioid receptor. Furthermore, the enhancement of the morphine-induced place preference in diabetic mice may be due to the up-regulation of delta-opioid receptor-mediated functions.

Animals↗

PubMed1997

Effect of water on lamellar structure of DPPC/sugar systems.

The ability of two monosaccharides, four disaccharides and one trisaccharide, to lower the transition temperature of L-alpha-dipalmitoyl phosphatidylcholine (DPPC) was investigated using differential scanning calorimetry (DSC) and the ability of these sugars to change the lateral packing of the acyl chains of DPPC was investigated using wide-angle X-ray diffraction. The sugars affected the gel-liquid crystal transition temperature (Tc) of DPPC when the water content of the DPPC/sugar systems was less than 20 wt.%. Specifically, Tc of the DPPC without sugar increased to approximately 106 degrees C, the Tc of the DPPC/monosaccharide system remained almost constant at 43 degrees C and of the DPPC/disaccharide or trisaccharide systems decreased to approximately 24 degrees C. In the dehydrated state, di- and trisaccharides caused looser packing of the DPPC hydrocarbon chains than the monosaccharides did, and the sugars affected the packing mode in different ways. The addition of water caused this difference in the sugars' effects on the packing mode to disappear and further addition of water caused the effect of sugar to almost disappear. Thus, the addition of water to a DPPC/sugar system weakens the interaction between the sugar and lipid and strengthens the DPPC chain packing.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

Binding Sites↗

PubMed1997

Mutational study of the amino-terminal domain of human tissue inhibitor of metalloproteinases 1 (TIMP-1) locates an inhibitory region for matrix metalloproteinases.

A bacterial expression system for the inhibitory N-terminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren, S. R., and Brew, K. (1996) FEBS Lett. 384, 155-161) has been used to produce 20 single- and double-site mutants that probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in the Ki for a C-terminally truncated form of stromelysin 1, MMP-3(DeltaC), but do not disturb the conformation involve substitutions of residues that are located in a ridge that is centered around the disulfide bond between Cys1 and Cys70. Specific residues that have a large influence on activity include Cys1, Thr2, Met66, Val69, and Cys70. Of the mutations introduced, the greatest functional disturbances, reflected in Ki increases of 2-4 orders of magnitude, are generated by changes that disrupt the Cys1-Cys70 disulfide bond and by substitution of Ala for Thr2. Most mutations that perturb the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A). However, the Thr2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that it is feasible to engineer TIMP-1 variants that are more specifically targeted to selected matrix metalloproteinases. The reactive site identified by these studies is a structurally constrained but elongated region of TIMP that can fit the matrix metalloproteinase substrate-binding site.

Binding Sites↗

PubMed1997

The role of delta-opioid receptors in the discriminative stimulus properties of a low dose of methamphetamine.

The effects of selective mu-, delta- and kappa-opioid receptor agonists and antagonists on the discriminative stimulus properties of methamphetamine were examined in rats that had been trained to discriminate between methamphetamine (0.4 mg/kg) and saline. Methamphetamine produced a dose-related increase in methamphetamine-appropriate responses in all of the rats. In generalization tests, neither morphine (a mu-opioid receptor agonist: 0.3-10 mg/kg) nor 3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexo]benzeneacetamide (U50,488H: a kappa-opioid receptor agonist: 1.0-8.0 mg/kg) generalized to the discriminative stimulus properties of methamphetamine. A newly synthesized non-peptide selective delta-opioid receptor agonist 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aalpha- octahydroquinolino(2,3,3,-g)isoquinoline (TAN-67: 32 mg/kg) partially generalized (70% methamphetamine-appropriate responses) to the discriminative stimulus properties of methamphetamine. In combination tests, pretreatment with the mu- and kappa-opioid receptor antagonists, beta-funaltrexamine (9.0 mg/kg) and nor-binaltorphimine (10 mg/kg), respectively, had little or no influence on the discriminative stimulus properties of methamphetamine. In contrast, pretreatment with naltrindole (a non-selective delta-opioid receptor antagonist: 3.0 mg/kg) or naltriben (a selective delta2-opioid receptor antagonist: 1.0 mg/kg), but not with 7-benzylidenenaltrexone (a selective delta1-opioid receptor antagonist: 0.5 and 1.0 mg/kg), significantly attenuated the discriminative stimulus properties of methamphetamine. However, naltrindole (3.0 mg/kg) did not significantly attenuate the discriminative stimulus properties of methamphetamine at a higher training dose (1.0 mg/kg). Our findings may have some bearing on the relative importance of the role of delta-opioid (especially delta2-opioid) receptors in the discriminative stimulus properties of a low dose of methamphetamine.

Animals↗

PubMed1997

Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells.

Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in 125I fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting. Urokinase was bound to its high affinity receptor on the vesicle membrane. Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation. We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion.

Blood↗

PubMed1997

Involvement of a region near valine-69 of tissue inhibitor of metalloproteinases (TIMP)-1 in the interaction with matrix metalloproteinase 3 (stromelysin 1).

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) by forming a 1:1 stoichiometric complex, but the inhibition mechanism of these inhibitors is not known. Here we have investigated the reactive site of TIMP-1 by its proteinase susceptibility before and after forming a complex with MMP-3 (stromelysin 1). When TIMP-1 was allowed to react with human neutrophil elastase, its inhibitory activity was destroyed. This resulted from cleavage of the Val69-Cys70 bond. However, cleavage of this bond by neutrophil elastase was prevented when TIMP-1 formed a complex with the catalytic domain of MMP-3, and full TIMP-1 activity was restored after dissociation of the complex at pH 3.0 in the presence of EDTA. These results indicate that the region around Val69 closely associates with an active MMP. The three-dimensional structure of the N-terminal domain of TIMP-2 elucidated by NMR studies [Williamson, Martorell, Carr, Murphy, Docherty, Freedman and Feeney (1994) Biochemistry 33, 11745-11759] reveals that Val69 and Cys70 form part of an extended ridge that also includes the N-terminal section of the inhibitor. This region is probably involved in the interaction with the catalytic domains of MMPs.

Binding Sites↗

PubMed1997

The role of spinal delta1-opioid receptors in inhibiting the formalin-induced nociceptive response in diabetic mice.

Injection of formalin into the hindpaw of mice produced a biphasic nociceptive response consisting of immediate (first-phase) and tonic (second-phase) components. In diabetic mice, the flinching response of the first phase was increased while that in the second phase was decreased in diabetic mice relative to the results in non-diabetic mice. To examine the role of supraspinal and/or spinal endogenous delta1-opioid receptors in inhibiting the formalin-induced nociceptive response in diabetic mice, we assessed the effect of 7-benzylidenenaltrexone, a selective delta1-opioid receptor antagonist, and naltriben, a selective delta2-opioid receptor antagonist, administered either i.c.v. or i.t., on the formalin-induced flinching response. The second-phase response appeared when diabetic mice were pretreated with 7-benzylidenenaltrexone (0.1 and 0.3 mg/kg, s.c.), but not with naltriben (0.3 and 1 mg/kg, s.c.). On the other hand, while 7-benzylidenenaltrexone (0.1, 0.3 and 1 microg/mouse) administered i.t. had no significant effect on the first phase, it significantly and dose-dependently increased the second phase of the formalin-induced flinching response in diabetic mice. 7-Benzylidenenaltrexone (1 and 3 microg/mouse) administered i.c.v. had no significant effect on either the first- or the second-phase response in both non-diabetic and diabetic mice. These results suggest that a spinal delta1-opioid receptor-mediated endogenous antinociceptive system may inhibit the formalin-induced second phase of the nociceptive response in diabetic mice.

Analgesics↗

PubMed1997

Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor.

The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.

Animals↗

PubMed1997

Differential effects of mu-, delta- and kappa-opioid receptor agonists on the discriminative stimulus properties of cocaine in rats.

The effects of selective mu-, delta- and kappa-opioid receptor agonists on the discriminative stimulus properties of cocaine were examined in rats trained to discriminate between cocaine (10 mg/kg) and saline. Cocaine produced a dose-related increase in cocaine-appropriate responses in all of the rats. In generalization tests, neither morphine (mu-opioid receptor agonist) nor N-methyl-N-7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-11-4-benzofu ranacetamide (U50,488H: kappa-opioid receptor agonist) generalized to the discriminative stimulus properties of cocaine. On the other hand, the newly synthesized non-peptide selective delta-opioid receptor agonist 2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino(2,3,3,-g)isoquinoline (TAN-67) partially generalized (56.7% cocaine-appropriate responses) to the discriminative stimulus properties of cocaine. Intracerebroventricular (i.c.v.) administration of [D-Ala2]deltorphin II (peptide delta 2-opioid receptor agonist) completely generalized, while neither [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO: mu-opioid receptor agonist) nor [D-Pen2,D-Pen5]enkephalin (DPDPE; delta 1-opioid receptor agonist) generalized to the discriminative stimulus properties of cocaine. These results suggest that the discriminative stimulus properties of cocaine may be partially mediated by delta-opioid (especially delta 2-opioid) receptors. In combination tests, pretreatment with morphine (3.0 mg/kg) and TAN-67 (3.0 and 10 mg/kg) significantly potentiated the discriminative stimulus properties cocaine. In contrast, pretreatment with U50,488H (2.0 and 4.0 mg/kg) scarcely shifted the discriminative stimulus properties of cocaine. Furthermore, the potentiating effect of 3.0 mg/kg morphine on the discriminative stimulus properties of cocaine was attenuated by 2.0 mg/kg U50,488H. In contrast, the potentiating effect of 10 mg/kg TAN-67 on the discriminative stimulus properties of cocaine was not reversed by either 2.0 or 4.0 mg/kg U50,488H. These results suggest that mu-, delta- and kappa-opioid receptor agonists modulate the discriminative stimulus properties of cocaine through different mechanisms, perhaps through different effects on the dopaminergic system.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1997

Supraspinal delta 1-opioid receptor-mediated antinociceptive properties of (-)-TAN-67 in diabetic mice.

The antinociceptive potencies of the enantiomorphs of TAN-67 (2-methyl-4-alpha alpha-(3-hydroxyphenyl)-1,2,3,4,4 a 5, 12, 12 a alpha-octahydroquinolino[2,3,3,-g]isoquinoline), (-)-TAN-67 and (+)-TAN-67, given intracerebroventricularly (i.c.v.) on the antinociceptive response were studied in streptozotocin-induced diabetic mice using the tail-flick test. (-)-TAN-67 at doses of 3-10 micrograms given i.c.v. produced dose-dependent inhibition of the tail-flick response in both non-diabetic and diabetic mice. The antinociceptive effect of (-)-TAN-67 in the tail-flick test in diabetic mice was greater than that in non-diabetic mice. The antinociceptive effect of (-)-TAN-67 was not antagonized by pretreatment with either beta-funaltrexamine, a selective mu-opioid receptor antagonist, or nor-binaltorphimine, a selective kappa-opioid receptor antagonist. When 7-benzylidenenaltrexone, a selective delta 1-opioid receptor antagonist, was administered 10 min before treatment with (-)-TAN-67, the antinociceptive effect of (-)-TAN-67 was significantly antagonized. However, naltriben, a selective delta 2-opioid receptor antagonist, had no significant effect on the antinociceptive effect of (-)-TAN-67. On the other hand, in the tail-flick test. (+)-TAN-67 at doses of 3-30 micrograms given i.c.v. did not produce dose-dependent inhibition of the tail-flick response in either non-diabetic or diabetic mice. In conclusion, (-)-TAN-67, but not its enantiomer (+)-TAN-67, produced an antinociceptive effect through the activation of delta 1-opioid receptors.

Analgesics↗

PubMed1997

The mitogen-activated protein kinase and JAK-STAT signaling pathways are required for an oncostatin M-responsive element-mediated activation of matrix metalloproteinase 1 gene expression.

Both astrocytes in the central nervous system and fibroblasts in somatic tissues are not only the major sources of extracellular matrix components but also of matrix metalloproteinases (MMPs), a family of enzymes directly involved in extracellular matrix breakdown. We have analyzed the regulation of the expression of MMPs and TIMPs (tissue inhibitors of metalloproteinases) in human primary astrocytes stimulated with oncostatin M (OSM) and other extracellular mediators in comparison with normal human dermal fibroblasts. It was found that OSM induced/enhanced transcription of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin 1) in astrocytes, and MMP-1, MMP-9 (gelatinase B), and TIMP-1 in fibroblasts. Analysis of the signal transduction leading to activation of the MMP-1 gene revealed the presence of an OSM-responsive element (OMRE) encompassing the AP-1 binding site and the signal transducer and activator of transcription (STAT) binding element, which mediate activation by OSM. OMRE is also present in the TIMP-1 gene promoter and, although there are some differences in these two motifs, both appear to be targets for the simultaneous action of OSM-induced nuclear effectors. The induced enhancement of transcription by synergistically acting AP-1 and STAT binding elements in response to OSM is Raf-dependent. Cross-talk between the mitogen-activated protein kinase and JAK-STAT pathways is required to achieve maximal induction of the OMRE-driven transcription by OSM.

Base Sequence↗

PubMed1997

Involvement of dopamine-dependent and -independent mechanisms in the rewarding effects mediated by delta opioid receptor subtypes in mice.

The rewarding effects of the delta 1 opioid receptor agonist [D-Pen2, Pen5]enkephalin (DPDPE) and the delta 2 opioid receptor agonist [D-Ala2]deltorphin II (DELT) on the activity of mesolimbic and nigrostriatal dopamine (DA) neurons were examined in mice. Both DPDPE (15 nmol, i.c.v.) and DELT (5 nmol, i.c.v.) produced a significant place preference in mice. The DPDPE (15 mol, i.c.v.)-induced place preference was abolished by 7-benzylidenenaltrexone (BNTX; 0.5 mg/kg, s.c.), a delta1 opioid receptor antagonist, but not by naltriben (NTB; 0.5 mg/kg, s.c.), a delta 2 opioid receptor antagonist. In contrast, the DELT (5 nmol, i.c.v.)-induced place preference was antagonized by NTB, but not BNTX. I.c.v.. injection of DPDPE, but not DELT, at a dose that produced a significant place preference produced a significant elevation of DA turnover in the mouse limbic forebrain, and this effect of DPDPE was antagonized by BNTX but not by NTB. In addition, i.c.v. injection of DPDPE or DELT not affect DA turnover in the mouse striatum. These results suggest that the rewarding effects produced by the activation of central delta 1, but not delta 2, opioid receptors may be caused through the enhancement of the mesolimbic DA neurotransmission, and confirm our previous hypothesis that the DA-dependent and -independent mechanisms may exist in the rewarding effects produced by the activation of central delta opioid receptor subtypes.

Analgesics↗

PubMed1997

Slowly conducting, low-threshold components of sensory nerve potentials in peripheral neuropathy: a microneurographic study.

By recording the averaged potential from a microelectrode inserted into the nerve, small late components of the sensory nerve action potential can be analyzed. We studied the thresholds of the late components in median nerves of 15 normal subjects and 37 patients with peripheral neuropathies. Under normal conditions, the late components reflected the activities of small myelinated fibers which had high thresholds. In the patients with peripheral neuropathies, a part of the late components often had abnormally low thresholds, occasionally the lowest. The presence of low-threshold, late components was related to the pathologies of the different types of neuropathy and correlated with the numbers of regenerating clusters in sural nerve biopsies. These slowly conducting, low-threshold components may originate from regenerating or remyelinating fibers, and therefore should have prognostic value.

Action Potentials↗

PubMed1997

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period.

The attachment of the blastocyst to the uterine luminal epithelium and the subsequent invasion by trophoblast cells through the stroma and deciduum occur in a highly regulated manner by remodeling of the extracellular matrix. We investigated the temporal and spatial expression of mRNAs for four matrix metalloproteinases (MMPs; MMP-2 [gelatinase A], MMP-3 [stromelysin 1], MMPs; MMP-2 [gelatinase B], and MMP-13 [collagenase 3]) and tissue inhibitors of metalloproteinases (TIMPs; TIMP-1, TIMP-2, and TIMP-3) in the mouse uterus from days 1 to 8 of pregnancy. Northern blot analyses showed the transcripts for MMP-2, MMP-3, RNA on these days. However, MMP-13 mRNA was not detected in the uterus, and only weak signals for MMP-3 mRNA were detected in the myometrium. Striking expression was observed with MMP-2 mRNA in the subepithelial stroma on days 3-5. With the progression of decidualization on day 6, signals were primarily in the secondary decidual zone. On day 8, MMP-2 mRNA was localized at the site of placenta formation in the mesometrial pole. Signals for MMP-9 mRNA were first detected in a small population of stromal cells exclusively at the site of implantation on day 5 at the antimesometrial pole. However, the most pronounced expressed was noted in trophoblast giant cells on day 8. TIMP-1 mRNA was present in the myometrium on day 1. On days 2-5, modest signals were detected in the stroma, and on days 6 and 8, they were in the secondary decidual zone. Localization of TIMP-2 mRNA was similar to that of TIMP-1 except it was restricted to the stroma on day 1. The regulation of TIMP-3 was more pronounced. While a gradual increase in signals was observed in stromal cells from days 1 to 4, strong signals were detected in antimesometrial stromal cells at the sites of blastocyst attachment on day 5. On days 6 and 7, even stronger signals were present in the primary decidual zone surrounding the embryo, and on day 8 signals were localized primarily in the mesometrial decidual bed. These results suggest that MMP-2 may participate in the early phase of decidualization and neovascularization required for placentation. The restricted MMP-9 expression in stromal cells on day 5 and in trophoblast giant cells on day 8, coupled with the expression of TIMP-3 in the stroma surrounding the embryo, suggests that a fine balance between MMP-9 and TIMP-3 may regulate trophoblast invasion in the uterus.

Animals↗

PubMed1997

Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-kappaB.

Interleukin-12 is produced in response to infection with bacteria or parasites or to bacterial constituents such as LPS in monocytes/macrophages and dendritic cells, and also generated by the interaction between activated T cells and antigen-presenting cells via CD40-CD40 ligand (CD40L). So far, transcriptional analyses of p40 have been carried out only using bacterial constituents such as LPS as stimuli. In the present study, we have characterized the transcriptional induction of p40 by CD40 ligation in a human B lymphoblastoid cell line, Daudi, and a human acute monocytic leukemia cell line, THP-1. These cells, stimulated by an agonistic monoclonal antibody against CD40 or by transfection with a CD40L expression vector, secreted p40 and showed enhanced p40 mRNA expression. Sequence analysis of the p40 promoter region identified two potential nuclear factor (NF)-kappaB binding sites conserved between mouse and human. Electrophoretic mobility shift assay revealed that the potential NF-kappaB binding sequence which is located around 120 bp upstream of the transcription initiation site in murine and human p40 genes formed an NF-kappaB complex with nuclear extract from Daudi cells stimulated by CD40 ligation. Moreover, transfection of Daudi cells with the polymerized NF-kappaB binding sequence ligated to a thymidine kinase/chloramphenicol acetyltransferase (CAT) reporter plasmid greatly induced CAT activity, but transfection with the polymerized mutated NF-kappaB binding sequence did not. These results suggest that the NF-kappaB binding site located around 120 bp upstream of the transcription initiation site in murine and human p40 promoter regions could be important for the p40 induction by CD40 ligation via activation of NF-kappaB.

Animals↗