L-Arginine β-naphthylamide hydrochloride

Bacteroides gingivalis is a Gram-negative micro-organism implicated in the pathogenesis of adult periodontitis and producing relatively large amounts of specific enzymes. In the present study, subgingival samples taken from adults with moderate periodontitis were examined for the presence and relative amounts of enzymatic activity toward certain substrates. Enzyme levels were then correlated with clinical periodontal indices and microbiological analysis of subgingival plaque, including darkfield microscopy for bacterial morphotypes and immunofluorescence microscopy for B. gingivalis and Bacteroides intermedius. The results of this study indicate a significant positive correlation between levels of enzyme capable of degrading N-benzoyl-D,L-arginine-beta-naphthylamide hydrochloride, and subgingival B. gingivalis (r = 0.55). There was a much lower correlation coefficient between this enzyme activity and subgingival B. intermedius (r = 0.26). Statistically significant (p less than 0.01) positive correlations were also demonstrated between total bacterial cell counts and levels of enzymatic activity against N-benzoyl-D,L-arginine-beta-naphthylamide hydrochloride (r = 0.76), N-carbobenzoxy-glycyl-glycyl-L-arginine-beta-naphthylamide hydrochloride (r = 0.72), and glycyl-L-proline-4-methoxy-beta-naphthylamide hydrochloride (r = 0.72), and glycyl-L-proline-4-methoxy-beta-naphthylamide hydrochloride (r = 0.69). There were significant differences in the levels of these three enzymatic activities between sites exhibiting various degrees of clinical severity of gingival inflammation and harboring various proportions of B. gingivalis. The data from this study indicate that measurement of specific enzymatic activities in subgingival samples can be useful in the diagnosis of B. gingivalis-associated periodontitis.

Background: Cysteine proteases (mainly cathepsins B and L) are thought to play an important role in the progress of cancer, including brain tumors. Together with other proteases, they hydrolyze the extracellular matrix and basement membrane proteins, thus enabling the tumor to grow and spread. Therefore cysteine protease inhibitors are regarded as protective factors, able to prevent tumor growth and dissemination. Material and methods: In this study, the activity of cysteine protease inhibitors (CPIs) was investigated in material derived from patients with brain tumors (astrocytoma and meningioma). The activity of CPIs was measured as antipapain activity in tissue homogenates, cerebrospinal fluid, and serum, with N-benzoyl-DL-arginine-2-naphthylamide hydrochloride (BANA) as a substrate, according to Barret's method. Results: Tumorous tissues showed higher activity of cysteine protease inhibitors than control tissues, but this difference proved to be statistically insignificant. The activity of CPIs was lower in cerebrospinal fluid and serum from patients with brain tumors. Conclusions: The activity of CPIs measured in brain tumor tissue cannot be taken as a marker of any type of tumor, whereas CPI activity in cerebrospinal fluid and serum may be considered a marker of meningioma. In meningioma patients the level of CPIs may be too low to prevent the host tissues from the growing tumor.

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca2+]i transient in a dose-dependent manner, measured by fura-2 fluorimetry and digital video imaging. The transient consisted of a rise and a decay and [Ca2+]i returned to baseline without oscillations. External Ca2+ influx occurred, as demonstrated by Mn2+ quench and external Ca2+ removal measurements. Bradykinin acted by stimulating bradykinin B2 receptors as evidenced by blockade by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl -3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolineca rbonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl-L-arginine (HOE 140) but not by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-proylglycyl- 3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecar bonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl ([Des-Arg]HOE 140). The [Ca2+]i signal was abolished by 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca2+ from internal stores since it was abolished by pretreatment with thapsigargin or cyclopiazonic acid. The mobilization of the internal Ca2+ store subsequently triggered a 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride (SKF 96365)-insensitive Ca2+ entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-beta-naphthylamide did not alter the transient, thus excluding the participation of mitochondria and lysosomes. Efflux via Ca2+ pumps contributed to the decay of the transient. Efflux via Na+/Ca2+ exchange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 12-myristate 13-acetate, and was enhanced by the protein kinase C inhibitors sphingosine and chelerythrine, the protein kinase A inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone, N-[2-(p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and agents that elevated levels of 3',5'-cyclic guanosine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP.