Minocycline and matrix metalloproteinase inhibition in acute intracerebral hemorrhage: a pilot study.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is a devastating cerebrovascular disorder with high morbidity and mortality. Minocycline is a matrix metalloproteinase-9 (MMP-9) inhibitor that may attenuate secondary mechanisms of injury in ICH. The feasibility and safety of minocycline in ICH patients were evaluated in a pilot, double-blinded, placebo-controlled randomized clinical trial. METHODS: Patients with acute onset (<12 h from symptom onset) ICH and small initial hematoma volume (<30 ml) were randomized to high-dose (10 mg/kg) intravenous minocycline or placebo. The outcome events included adverse events, change in serial National Institutes of Health Stroke Scale score assessments, hematoma volume and MMP-9 measurements, 3-month functional outcome (modified Rankin score) and mortality. RESULTS: A total of 20 patients were randomized to minocycline (n = 10) or placebo (n = 10). The two groups did not differ in terms of baseline characteristics. No serious adverse events or complications were noted with minocycline infusion. The two groups did not differ in any of the clinical and radiological outcomes. Day 5 serum MMP-9 levels tended to be lower in the minocycline group (372 ± 216 ng/ml vs. 472 ± 235 ng/ml; P = 0.052). Multiple linear regression analysis showed that minocycline was associated with a 217.65 (95% confidence interval -425.21 to -10.10, P = 0.041) decrease in MMP-9 levels between days 1 and 5. CONCLUSIONS: High-dose intravenous minocycline can be safely administered to patients with ICH. Larger randomized clinical trials evaluating the efficacy of minocycline and MMP-9 inhibition in ICH patients are required.

Angiotensin type 1 receptor A1166C polymorphism and systemic lupus erythematosus: correlation with cellular immunity and oxidative stress markers.

Angiotensin II, one of the rennin-angiotensin system components, is important in the cardiovascular hemodynamic and plays an important role in the development of cardiovascular disease in systemic lupus erythematosus (SLE) patients. The angiotensin II, through interaction with angiotensin II type 1 receptor (AGTR1), promotes proliferation, inflammation and fibrosis. The single nucleotide polymorphism of the AGTR1 (dbSNP: rs5186) gene can be associated with development and progression of SLE disease. The aims of this study were to compare the frequency of AGTR1 rs5186 in SLE patients with healthy individuals and to evaluate possible association between AGTR1 A1166C gene polymorphism and serum level of lipids, neopterin and malondialdehyde in SLE patients from a population of West Iran. One hundred SLE patients and 98 healthy subjects were studied. The AGTR1 A1166C polymorphism was detected by polymerase chain reaction- restriction fragment length polymorphism method and the serum lipid profile was obtained by enzymatic method. Neopterin and malondialdehyde were detected using high-performance liquid chromatography. We did not detect significant association between AGTR1 A1166C polymorphism and the risk of SLE. The levels of triglyceride (225 ± 118 mg/dl), neopterin (30 ± 24 nmol/l) and malondialdehyde (25 ± 9.6 nmol/l) in SLE patients were significantly higher than those in control subjects (139 ± 56 mg/dl, p = 0.03, 6.4 ± 2, p = 0.03, 9.4 ± 2.5 nmol/l, p = 0.01, respectively). Individuals with AGTR1 AC + CC genotype had higher levels of total cholesterol and malondialdehyde compared with those with AGTR1 AA genotype. SLE patients with either AGTR1 AA or AGTR1AC + CC genotype had significantly higher malondialdehyde or neopterin levels compared with the corresponding control subjects. In conclusion, although the present study did not find any association between AGTR1 A1166C polymorphism and the risk of SLE, the presence of this polymorphism was associated with higher levels of malondialdehyde and higher concentration of neopterin in patients.

Association between the cytotoxic T-lymphocyte antigen-4 mutations and the susceptibility to systemic lupus erythematosus; Contribution markers of inflammation and oxidative stress.

The cytotoxic T lymphocyte antigen-4 (CTLA-4) also known as CD152 (cluster of differentiation 152) is a crucial negative regulator of the immune system. This protein receptor provides negative signals in order to suppress T-cell activation and immune attack against self-antigens, although its role is unclear.  The ability of CTLA-4 to limit T cell-mediated immune response has made it a major target in treatment of tumors and autoimmune diseases such as systemic lupus erythematosus (SLE). In this study, we investigated whether CTLA-4 G-1661A and CTLA-4 T-1722C mutations are associated with SLE. So one hundred nine SLE patients and 101 gender and age-matched unrelated healthy controls were recruited for this case-control study. The promoter mutations were detected by PCR-RFLP, neopterin, malondialdehyde (MDA) and serum lipid concentration were determined by HPLC and enzyme assay, respectively. RESULT: We found that both codominant (AA vs. GG) and recessive (AA vs. GA+GG) CTLA-4 G-1661A mutation significantly decreased the risk of SLE by 1.7 and 3.7 times, respectively.  Interestingly, SLE patients with AA genotypes of CTLA-4 G-1661A have lower neopterin and MDA concentration compared with GA+GG genotypes. The overall distribution of CTLA-4 T-1722C genotypes and alleles in SLE patients were similar to those in control group. In conclusion, our findings showed, that there is an association between systemic inflammatory markers, oxidative stress and the CTLA-4 G-1661A GG+AG genotypes, MDA and neopterin which are the most conventional risk factors for coronary heart disease, therefore these mutations may be consider as a risk factor for susceptibility to heart disease in SLE patients.

Pharmacogenetics of drug metabolizing enzyme: thiopurine methyl transferase phenotypes and multidrug resistance 1 gene polymorphism in inflammatory bowel disease.

Inflammatory bowel disease(IBD) is progressing rapidly in developing countries such as Iran. This research is intended to compile the frequency distribution of the drug metabolizing enzyme, thiopurine methyl transferase(TPMT) and the drug transporter, Multi drug resistance(MDR1) which are involved in metabolism of many therapeutics such as thiopurines in inflammatory bowel disease(IBD). Ethnicity is an important variable influencing drug response. The aims of this research were to investigate the association of TPMT phenotypes with MDR1 genotypes. TPMT activity was measured by using a non-extraction HPLC method and genotype for the C3435T polymorphism of MDR1 gene was determined in 215 unrelated IBD patients including of 85 males and 130 females and 212 unrelated healthy individuals consisted of 96 males and 116 females as control group by PCR-RFLP in Iran's western population. TPMT phenotypes demonstrated no frequency for deficient, 2.2% for low and 97.8% for normal activity that is different with results of other studies. Interestingly there were a significant negative correlation between TPMT activities as calculated based on nmol/grHb/h and positive correlation calculated in mU/L with Hb levels in IBD patients and control subjects. Dominant and codominant MDR1 C3435T gene polymorphism increased the risk of IBD by 1.45 and 1.46 times, respectively. IBD patients with MDR1 mutant genotypes C3435T, had lower TPMT activites and Hb concentrations. Using of mU/L is more appropriate than nmol6MTG/grHb/h for expressing TPMT activity. TPMT frequency of deficient and low activity in western Iran is low. The carriers of mutant C3435T MDR1 are not good TPMT methylators.

Matrix metalloproteinase 9 polymorphisms and systemic lupus erythematosus: correlation with systemic inflammatory markers and oxidative stress.

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs and is characterized by persistent systemic inflammation. Among the effects of inflammatory mediators, the induction of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and oxidative stress has been demonstrated to be important in the development of SLE. In this study, the possible association between MMP-9 and MMP-2 functional promoter polymorphism, stress, and inflammatory markers with development of severe cardiovascular disease (CVD), high blood pressure (HBP), and lupus nephropathy (LN) in SLE patients was investigated. The present case-control study consisted of 109 SLE patients with and without CVD, HBP and LN and 101 gender- and age-matched unrelated healthy controls from a population in western Iran. MMP-2 -G1575A and MMP-9 -C1562T polymorphisms were detected by PCR-RFLP, serum MMP-2 and MMP-9, neopterin, malondialdehyde (MDA) and lipid levels were determined by ELISA, HPLC and enzyme assay, respectively. We found that MMP-9 -C1562 T and MMP-2 -G1575A alleles act synergistically to increase the risk of SLE by 2.98 times (p = 0.015). Findings of this study also demonstrated that there is a significant increase in the serum levels of MMP-2, neopterin and MDA and a significant decrease in serum level of MMP-9 in the presence of MMP-9-C1562 T and MMP-2 -G1575A alleles in SLE patients compared to controls. Further, SLE patients with MMP-9 (C/T + T/T) genotype had significantly higher serum concentrations of MMP-2, neopterin, MDA and LDL-C, but lower serum MMP-9 and HDL-C levels than corresponding members of the control group. MMP-9 (C/T + T/T) genotype increased risk of hypertension in SLE patients 2.71-fold. This study for the first time not only suggests that MMP-9 -C1562 T and MMP-2 -G1575A alleles synergistically increase the risk of SLE but also high serum levels of MDA, neopterin, and circulatory levels of MMP-2 and lower MMP-9 in SLE patients. This information may be important in the evaluation of SLE progression and in the elucidation of the mechanisms of the disease pathogenesis.

Effect of central muscarinic receptors on passive-avoidance learning deficits induced by prenatal pentylenetetrazol kindling in male offspring.

Occurrence of the epileptic seizures during gestation might affect the neurodevelopment of the fetus resulting in cognitive problems for the child later in life. We have previously reported that prenatal pentylenetetrazol (PTZ)-kindling induces learning and memory deficits in the children born to kindled mothers, later in life but the mechanisms involved in this processes are unknown. The cholinergic system plays a major role in learning and memory. The present study was performed to investigate the possible involvement of central muscarinic cholinergic receptors on learning and memory deficits induced by prenatal PTZ-kindling in male offspring. Pregnant Wistar rats were kindled by repetitive i.p. injection of 25mg/kg of PTZ on day 13 of their pregnancy. The effect of intracerebroventricular (ICV) microinjection of scopolamine and pilocarpine, muscarinic cholinergic receptors antagonist and agonist, respectively on passive-avoidance learning of pups were tested at 12weeks of age using shuttle-box apparatus. Our data showed that the retention latencies of pups that received scopolamine (2 or 3µg) were significantly reduced compared to those received normal saline (p<0.05). Interestingly, post training ICV administration of pilocarpine (2µg) retrieved pups' memory deficits (p<0.001). These results demonstrate for the first time, the importance of the central muscarinic cholinergic receptors in learning and memory deficits in pups born to kindled dams and suggest a central mechanism for the cognitive and memory dysfunction, associated with seizures during pregnancy.

Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report.

There is some evidence indicating lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and glomerulonephritis in systemic lupus erythematosus (SLE) patients. Human butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are two major bioscavenger enzymes that are associated with inflammation, oxidative stress and lipid metabolism. Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVDs in SLE. It has also been suggested that deficiency in the function of the antioxidant system and an increase in reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE. This study is the first investigation to examine the association of BuChE phenotypes, PON1 (L55M; PON-55-M) polymorphism, the levels of malondialdehyde (MDA), neopterin, lipid-lipoprotein and activities of BuChE and arylesterase activity (ARE) of PON with severity of SLE. The present case-control study consisted of 109 SLE patients and 101 gender- and age-matched, unrelated healthy control subjects from the population of west Iran. We found that the PON-55-M allele and BuChE non-UU act synergistically to increase the risk of SLE by 2.5 times (1.03-6.7, p = 0.044). There was a significant negative correlation between severity of SLE with serum BuChE activity (R = -0.31, p < 0.001) and positive correlation with serum neopterin level. The SLE patients with the PON-55-M (M/L + M/M) allele or with BuChE non-UU phenotype had significantly lower serum ARE and BuChE activities than those with PON-55-L/L or BuChE-UU phenotypes, respectively. In addition, their serum levels of MDA, neopterin and LDL-C were significantly elevated, suggesting that these individuals are more susceptible to CVD. However, further studies are needed to shed more light on the contribution of the M allele of PON1 and non-UU phenotypes of BuChE in the development of SLE in different ethnicities.

Methylentetrahydrofolatereductase (rs1801133) polymorphism and psoriasis: contribution to oxidative stress, lipid peroxidation and correlation with vascular adhesion protein 1, preliminary report.

BACKGROUND: Psoriatic patients are at greater risk of oxidative stress and inflammation which is associated with abnormal plasma lipid metabolism and lipid peroxidation.There is not any information about the clinical significance of relation between methylentetrahydrofolatereductase (MTHFR) 677-T allele with malondialdehyde (MDA), lipids, apolipoproteins and vascular adhesion protein-1 (VAP-1) partakes in the migration process of lymphocytes into sites of inflammation. OBJECTIVES: This study is the first investigation to examine the association of MTHFR (rs1801133) C677T polymorphism, serum level of MDA, VAP-1, lipid-lipoprotein and apolipoproteins with the risk of psoriasis. METHODS: The present case-control study consisted of 100 psoriatic patients and 100 gender- and age-matched unrelated healthy controls from west population of Iran. MTHFR-C677T (rs1801133) polymorphisms were detected by restriction fragment length polymorphism (PCR-RFLP), VAP-1 by ELISA, apolipoproteins by immunoprecipitation, lipid and apolipoproteins by spectrophotometery and MDA by HPLC. RESULTS: We found that dominant/recessive model (CC + CT/TT) and T allele of MTHFR-677 alleles significantly 7.45 and 1.76 times increased risk of psoriasis, respectively. The psoriasis patients with MTHFR-677-T (C/T + T/T) allele had significantly higher serum MDA, VAP-1 and apolipoproteinsAPOB concentrations and ratio of APOB/APOA1 than the control subjects.The MTHFR-677-T allele frequencies in psoriasis patients were significantly higher than that in control group (28.5% vs. 18.5%; P = 0.018).We found a significant positive correlation between VAP-1 with MDA (P = 0.047) and LP (a) (P = 0.025). CONCLUSION: In the present study, we demonstrated for the first time that the psoriasis patients with MTHFR-677-T (C/T + T/T) allele had higher serum levels of MDA, VAP-1, APOB and ratio of APOB/APOA1 and dominant/recessive model (CC+CT/TT) and T allele of MTHFR-677 are significantly more common in psoriasis and increased risk of psoriasis by 7.45 and 1.76 fold, respectively. These data suggest that psoriasis patients carrying of TT genotypes and T allele of MTHFR-677 may be more susceptible to cardiovascular disease and myocardial infarction.

Paraoxonase 1 (PON1) 55 polymorphism, lipid profiles and psoriasis.

BACKGROUND: Paraoxonase 1 (PON1) is a serum high-density lipoprotein-bound enzyme with antioxidant function. It hydrolyses lipid peroxides, protecting low-density lipoproteins from oxidative modifications. Patients with psoriasis are at greater risk of oxidative stress, which is associated with abnormal plasma lipid metabolism. OBJECTIVES: In this study, association of the PON1 55 M allele with serum arylesterase (ARE) activity, malondialdehyde (MDA), lipid profiles and psoriasis was investigated. METHODS: The present case-control study consisted of 100 patients with psoriasis with and without cardiovascular diseases (mean age 35·3 years) and 100 sex- and age-matched unrelated healthy controls (mean age 35·7 years) from the population of western Iran. The PON1 55 Met>Leu polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism. Serum ARE activity, MDA, and lipid and apolipoprotein levels were determined spectrophotometrically, by high-performance liquid chromatography and by enzyme assay, respectively. RESULTS: The presence of the PON1 55 M allele was found to be associated with psoriasis (odds ratio = 1·96, P = 0·017). The patients with psoriasis with the PON1 M (M/L + M/M) allele had higher MDA levels (4·12 ± 0·88 vs. 2·24 ± 0·55 µmol L(-1) , P < 0·001), apolipoprotein B (APOB)/APOA1 ratio (0·91 ± 0·66 vs. 0·66 ± 0·35, P = 0·004), APOB (111 ± 38·7 vs. 88·3 ± 22·5 mg mL(-1) , P = 0·001) and lipoprotein(a) [LP(a)] (21·9 ± 18·4 vs. 15·8 ± 16·6 mg mL(-1) , P = 0·034), but lower ARE activity (39·6 ± 11 vs. 45·9 ± 11·8 U mL(-1) , P = 0·031) than the control subjects. ARE activity showed a significant positive correlation with APOA1 and a negative correlation with MDA concentration in patients with psoriasis. CONCLUSIONS: The PON1 55 M allele is a risk factor for psoriasis. Carriers of this allele have high levels of MDA, APOB and LP(a), a high APOB/APOA1 ratio and low ARE activity. These results indicate that oxidative stress, impairment of the antioxidant system and abnormal lipid metabolism may play a role in the pathogenesis and progression of psoriasis and its related complications. These data suggest that patients with psoriasis are more susceptible to vascular diseases.

Matrix metalloproteinase-2 functional promoter polymorphism G1575A is associated with elevated circulatory MMP-2 levels and increased risk of cardiovascular disease in systemic lupus erythematosus patients.

Matrix metalloproteinase-2 (MMP-2) is a zinc dependent endonuclease that degrades type IV collagen, the major structural component of basement membranes. MMP-2 functional promoter polymorphism G1575A affects circulating level of MMP-2 and may be considered an important genetic determinant of cardiovascular disease (CVD) in systemic lupus erythematosus (SLE) patients. In this study, association between MMP-2 1575A allele with serum MMP-2, neopterin and lipid-lipoprotein levels and with SLE and developing CVD was investigated. The present case-control study consisted of 109 SLE patients with and without CVD (mean age, 35.6 years) and 101 gender- and age-matched, unrelated, healthy controls (mean age, 37.1 years) from the population in the west of Iran. MMP-2 1575G/A polymorphism was detected by polymerase chain reaction (restriction fragment length polymorphism) PCR-RFLP, serum MMP-2, neopterin and lipid levels were determined by enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and enzyme assay, respectively. The presence of MMP-2 G1575A allele was found to be associated with SLE and developed CVD (OR = 1.78, p = 0.029 and OR = 3.43, p = 0.025, respectively). The SLE patients with MMP-2 A (G/A + A/A) allele had higher MMP-2 activity (301 ± 166 vs. 194 ± 35.5, p = 0.002), neopterin (29.4 ± 39.4 vs. 7.3 ± 4.6, p = 0.005), LDL-C (120 ± 25.7 vs. 87 ± 39.3, p = 0.045) and lower HDL-C (39.6 ± 11 vs. 45.9 ± 11.8, p = 0.031) levels than the control subjects. There was a significantly positive correlation between MMP-2 level with neopterin, total cholesterol and TG levels and negative correlation with HDL-C level in SLE patients with CVD. MMP-2 G1575A allele may be a risk factor for SLE. The carriers of this allele have high levels of MMP-2, neopterin, total cholesterol and TG and lower levels of HDL, thus, they are more likely to develop heart disease.

Effect of prenatal pentylenetetrazol-induced kindling on learning and memory of male offspring.

Current data concerning the effects of maternal seizure during pregnancy on newborns are limited. This study was carried out to investigate the effect of prenatal pentylenetetrazol (PTZ)-induced kindling on learning and memory of offspring. Female Wistar rats were kindled with i.p. injections of 25 mg/kg of PTZ on day 13 of their pregnancy. The spatial performance and passive avoidance learning of pups were tested at 7 weeks and 12 weeks of age using Morris water maze (MWM) task and shuttle-box apparatus, respectively. We found, for the first time, that prenatal exposure to maternal seizure induced by PTZ leads to a significant impairment of learning and memory. In addition, the number of live birth was significantly lower in kindled rats compared to control. In MWM studies, the young offspring of kindled rats had poor spatial learning ability. The frequent tonic-clonic seizures in pregnancy was also associated with a poor memory as evidenced by decrease in distance swam in the target quadrant by the offspring of the kindled mother in the adulthood. Data obtained from shuttle-box studies showed that retention latencies of pups born to kindled dams were significantly reduced compared to those born to control dams. The hippocampus, amygdala and frontal cortex are very important for memory consolidation and our data suggest that subsequent developmental events are not sufficient to overcome the adverse effects of prenatal exposure to maternal seizures to these regions of the brain. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy during pregnancy.

Butyrylcholinesterase K variants increase the risk of coronary artery disease in the population of western Iran.

The conflicting results of several studies suggest that there is an association between the butyrylcholinesterase-K variant (BCHE-K, G1615A/Ala539Thr) and the risk of developing coronary artery disease (CAD) in diabetes and non-diabetic subjects. The objective of this study was to determine whether the presence of the BCHE-K variant exacerbates the risk of CAD in patients from western Iran with and without type 2 diabetes mellitus (T2DM). This case-control study comprised 464 subjects undergoing their first coronary angiography. They were matched and randomly assigned into four groups: CAD+T2DM+ (CAD/T2DM), CAD+DM(-) (CAD/ND), CAD(-)DM+ (T2DM/NCAD) and CAD(-)DM(-)(control). The BCHE-K variant was detected by PCR-RFLP. The BCHE-K allele frequency in CAD patients with and without T2DM [total CAD (TCAD)] and separately for each group (CAD/T2DM and CAD/ND) was significantly higher than in the control group (21.1 % versus 13.3 % (p = 0.001), 22.4 % versus 13.3 % (p = 0.001) and 19.7 % versus 13.3 % (p = 0.015), respectively). The odds ratios (ORs) for the BCHE-K heterozygous and homozygous variants in TCAD subjects were 1.65 (95 % CI 1.17-2.3; p = 0.004) and 4.3 (1.05-19.4; p = 0.048); for CAD/T2DM individuals 1.76 (1.2-2.6; p = 0.004) and 4.73 (0.96-23.3; p = 0.052); and for CAD/ND patients 1.53 (1.05-2.3; p = 0.029) and 3.88 (0.8-19.7; p = 0.7), respectively. The OR of the BCHE-K allele was found to be 1.74 (1.1-2.4; p = 0.001) in TCAD subjects, 1.87 (1.12-1.48; p = 0.001) in the CAD/T2DM group and 1.59 (1.04-1.4; p = 0.016) in CAD/ND subjects. These data suggest that the BCHE-K allele increases the risk of CAD in the population (with and without DM) in western parts of Iran, and its presence intensifies the risk of CAD in T2DM. The fact that the BCHE-K allele, even in the heterozygous form, exacerbates the risk of CAD in this population, suggests that a specific therapeutic intervention should be considered for this particular group of patients.

Association between apolipoprotein E polymorphism and coronary artery disease in the Kermanshah population in Iran.

OBJECTIVE: Coronary artery disease (CAD) is the major cause of death in developing countries, such as Iran. The apolipoprotein E gene (APOE) is considered an important genetic determinant of CAD. In this study, the relationship between APOE polymorphism with lipid variation in CAD patients in Kermanshah, Iran was investigated. METHODS: This case-control study consisted of 115 CAD patients who angiographically had at least 30% stenosis and 135 unrelated controls. APOE polymorphism was detected by PCR-RFLP and serum lipid level was measured enzymatically. RESULTS: The APOE-epsilon4 and -epsilon2 allele frequencies were significantly higher in the CAD patients than in the control group (P < 0.001). The CAD patients with epsilon3/epsilon4 genotype had also higher TC (P < 0.001) and LDL-C (P < 0.01) and lower HDL-C (P < 0.03) levels than that of the control group. CONCLUSIONS: APOE-epsilon4 allele is a risk factor for CAD, so that carriers of this allele with high levels of LDL-C may be susceptible to CAD and myocardial infarction.

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B.

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.

The fibronectin-like domain is required for the type V and XI collagenolytic activity of gelatinase B.

Gelatinase B (matrix metalloproteinase-9) is able to degrade several extracellular matrix proteins, including gelatin, elastin, and collagen types IV, V, XI, and XIV. This enzyme contains a "fibronectin-like" domain which is composed of three tandem copies of a fibronectin type 2 homology unit inserted into its catalytic domain. We have studied the involvement of this domain in the substrate specificity of gelatinase B by expressing a mutant of the enzyme, in Escherichia coli, in which this domain has been deleted. This mutant enzyme retained its ability to cleave the peptide substrate Mca-PLGL(Dpa)AR-NH2, possessing K(m) and kcat values similar to those of the wild-type enzyme. In addition, the NH2-terminal, 14-kDa, inhibitory domain of recombinant tissue inhibitor of metalloproteinase-2 was able to inhibit the mutant and the wild-type enzymes with the same potency. The mutant's gelatinolytic activity was also retained but reduced in comparison to that of the wild-type enzyme. However, contrary to the wild-type enzyme, the mutant was not able to digest or bind fibrillar collagen types V and XI. These data indicate that the fibronectin-like domain of gelatinase B is an important determinant of the enzyme's fibrillar collagen substrate specificity. It allows the enzyme to bind to and cleave collagen types V and XI, events which are thought to be involved in several normal physiological and pathological processes such as metastasis and arthritis.

Basic calcium phosphate crystals induce synthesis and secretion of 92 kDa gelatinase (gelatinase B/matrix metalloprotease 9) in human fibroblasts.

OBJECTIVE: Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe joint degeneration accompanied by synovial hypertrophy. The metalloprotease 92 kDa gelatinase (MMP-9) has been implicated in the degradation of extracellular matrix in osteoarthritis, but the ability of BCP crystals to induce gelatinase in human fibroblasts or in adult porcine chondrocytes has not previously been studied. The hypothesis that the mitogenic response to BCP crystals is accompanied by induction and secretion of MMP-9 was studied. METHODS: MMP-9 messenger RNA (mRNA) was detected by northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Gelatinase secretion was identified by western blot and zymography of conditioned media. RESULTS: BCP crystals caused a concentration dependent induction of MMP-9 mRNA accumulation and protein secretion in human fibroblasts but not in adult porcine chondrocytes. CONCLUSION: BCP crystals induce MMP-9 production by HF but not adult porcine chondrocytes. Fibroblast MMP-9 may be an important mediator of the joint destruction associated with synovial fluid BCP crystals.

Does muscle tension reflect arousal? Relationship between electromyographic and electroencephalographic recordings.

Increased muscle tension and heightened arousal are the most consistent finding in patients with anxiety disorders. This study examined the relationship between frontalis and gastrocnemius electromyographic (EMG) and electroencephalographic activity on 14 female generalized anxiety disorder patients and 14 female control subjects. In GAD patients, gastrocnemius but not frontalis EMG was correlated with right but not left hemisphere activity. For the non-anxious subjects, there was a pattern for both beta 1 and beta 2 waves to be positively associated with both frontalis and gastrocnemius muscle tension levels. The results, while preliminary, suggest that EMG activity may reflect central nervous system arousal.

Treatment, discontinuation, and psychomotor effects of diazepam in women with generalized anxiety disorder.

Twenty-one women with generalized anxiety disorder (GAD) participated in a 6-week, double-blind, placebo-controlled trial to assess the treatment and abrupt withdrawal effects of diazepam on psychic and somatic symptoms of anxiety. The results confirmed those of previous studies reporting that (1) clinical doses of diazepam are effective in attenuating the symptoms of generalized anxiety to a greater extent than placebo during the first 3 weeks of treatment; (2) somatic symptoms are more responsive to diazepam treatment than psychic symptoms; and (3) patients taking diazepam exhibit increased anxiety upon abrupt withdrawal of medication. This finding, combined with the fact that diazepam discontinuation did not produce withdrawal effects in non-anxious volunteers, suggests that diazepam discontinuation after 6 weeks results in rebound anxiety rather than a physical withdrawal syndrome. Diazepam did not improve psychomotor performance in GAD patients. Psychomotor impairment after 6 weeks of diazepam was similar to that seen in nonanxious volunteers.

Mechanism of Ca2+-dependent activity of human neutrophil gelatinase B.

Progelatinase B can be activated in vitro by organomercurial compounds and by proteolytic enzymes such as trypsin, chymotrypsin, and stromelysin. Activation of the proenzyme by either 4-aminophenylmercuric acetate or chymotrypsin yielded proteins that absolutely required Ca2+ for activity, regardless of the pH of the reaction mixture. The trypsin- and stromelysin-activated gelatinases, on the other hand, did not require Ca2+ for activity at pH 7.5, but the activity of the trypsin-activated enzyme became Ca2+ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent pKa of 8.8 was involved in this process. The NH2-terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH2 termini (Phe88), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met75 and Gln89 or Glu92 as the NH2-terminal amino acid, respectively. These data, in conjunction with the x-ray crystal structure of collagenase, suggest that a salt linkage involving Phe88 is responsible for the Ca2+-independent activity of trypsin- and stromelysin-activated gelatinase. Replacing Asp432 in progelatinase with either Glu, Asn, Gly, or Lys resulted in the proteins that, upon activation by trypsin, required Ca2+ for activity. These substitutions did not significantly affect Km for the synthetic substrate but decreased the kcat and increased the half-maximal Ca2+ concentration required for enzyme activity (KCa) by severalfold. The effects on kcat and KCa depended on both charge and size of the side chains of the substituted amino acids. The decrease in kcat correlated well with the increase in KCa of the mutants. The orders of decrease in kcat and increase in KCa were wild type >/= D432E > D432N > D432G > D432K and wild type