Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

Recombinant expression of human mast cell proteases chymase and tryptase.

Expression of recombinant human chymase and tryptase was achieved in a baculovirus-insect cell system using a fusion protein construct. Recombinant baculovirus was produced with DNA coding for a NH2-ubiquitin-chymase-COOH or NH2-ubiquitin-tryptase-COOH fusion protein inserted immediately downstream of the signal sequence for the secreted envelope protein, glycoprotein 67. In each construct, the natural prepropeptide sequence of the protease was replaced by the amino acid sequence for the enterokinase cleavage site of trypsinogen. High Five insect cells infected with either of the modified baculovirus produced mg quantities of each fusion protein per liter of culture. Treatment of the chymase-fusion protein with enterokinase or the tryptase-fusion protein with enterokinase in the presence of a highly charged polysaccharide (dextran sulfate or heparin) produced enzymatically active proteases with properties of the native enzymes. A procedure for the purification of mg quantities of recombinant chymase from infected-cell medium is presented.

Diverse effects of pH on the inhibition of human chymase by serpins.

Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.

Structural changes associated with the spontaneous inactivation of the serine proteinase human tryptase.

Human skin tryptase, a serine proteinase stored within mast cell secretory granules, rapidly loses enzymatic activity in solutions of physiological salt concentration, pH, and temperature. The inactivation of tryptase can be slowed and even reversed by addition of heparin, a highly sulfated glycosaminoglycan also found in the secretory granules. These properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underlying the functional instability of tryptase, circular dichroism (CD) and analytical ultracentrifugation were used to investigate structural changes during spontaneous inactivation. The CD spectra of active and spontaneously inactivated tryptase are different, particularly in the region around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotrypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activity resulting from spontaneous inactivation was accompanied by a diminution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activity loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of decay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase. The return of the CD signal appeared to be more rapid than the return of enzymatic activity, thereby suggesting the presence of an unidentified step which is rate-limiting for activity return (and loss) and subsequent (prior) to the CD change accompanying activity loss. Ultracentrifugation analysis of tryptase showed a marked change in its association state upon inactivation. Sedimentation equilibrium under stabilizing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mixture of species was evident, which was characterized as monomers and tetramers in equilibrium. These results demonstrate that spontaneous inactivation of tryptase is associated with reversible conformational changes and that a consequence of inactivation is the formation of a destabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tryptase and chymotrypsin.

Use of the polymerase chain reaction for the detection of Chlamydia trachomatis in clinical specimens and its comparison to commercially available tests.

A polymerase chain reaction (PCR) was developed to detect Chlamydia trachomatis in genital tract specimens. Two sets of primers for the PCR were used; one set amplifies a region of the plasmid present in all C trachomatis strains and the other amplifies a conserved region of the genome coding for the major outer membrane protein. The sensitivity of these PCRs were compared with each other, and with the sensitivities of antigen ELISA, Clearview and culture. Southern blotting and probing was used to increase sensitivity of detection.

Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin.

The physiological alpha-oxoaldehyde methylglyoxal binds and modifies arginine, lysine, and cysteine residues in proteins. The kinetics and mechanism of these reactions were investigated with N alpha-acetylamino acids and bovine serum albumin at pH 7.4 and 37 degrees C. The reaction of methylglyoxal with N alpha-acetylarginine involved the initial reversible formation of glycosylamine and 4,5-dihydroxy-5-methylimidazolidine derivatives, with further slow irreversible conversion to an imidazolone, N alpha-acetyl-N delta- (5-methyl-4-imidazolon-2-yl)ornithine. The imidazolone was fluorescent with an excitation lambda max value of 320 nm and an emission lambda max value of 398 nm. Methylglyoxal reacted reversibly with N alpha-acetyllysine to form glycosylamine and bisglycosylamine derivatives. Further reaction of these glycosylamines occurred to form brown, fluorescent oligomers that were not characterized. Methylglyoxal reacted rapidly and reversibly with N alpha-acetylcysteine to form the hemithioacetal adduct. The reaction of methylglyoxal with bovine serum albumin (BSA) at pH 7.4 and 37 degrees C involved the reversible and irreversible formation of methylglyoxal-BSA adducts. Irreversible modification of BSA occurred mainly on arginine residues to form imidazolone. The formation of methylglyoxal-modified proteins involves glycoxidation leading to advanced glycation end product-like fluorescence. It is expected to be increased in diabetes mellitus and may be linked to the development of diabetic complications.

The reaction of methylglyoxal with aminoguanidine under physiological conditions and prevention of methylglyoxal binding to plasma proteins.

Increased formation of methylglyoxal in clinical diabetes mellitus and metabolism by the glyoxalase system has been linked to the development of clinical complications of diabetes: retinopathy, neuropathy and nephropathy. Aminoguanidine has been proposed as a prophylactic agent for preventive therapy of diabetic complications. Methylglyoxal reacted with aminoguanidine under physiological conditions to form two isomeric triazines, 3-amino-5-methyl-1,2,4-triazine and 3-amino-6-methyl-1,2,4-triazine. The mean second order rate constant for the reaction of methylglyoxal with aminoguanidine, kMG.AG = 0.39 +/- 0.06 M-1 sec-1 at pH 7.4 and 37 degrees. Under these conditions, no methylglyoxal bisguanylhydrazone was detected. Aminoguanidine prevented the irreversible modification of human plasma protein by a physiological concentration of methylglyoxal (1 microM); the median inhibitory concentration IC50 value of aminoguanidine was 203 +/- 16 microM (N = 28). The scavenging of methylglyoxal by aminoguanidine may contribute to the beneficial effects of aminoguanidine in the prevention of vascular pathogenesis in diabetes.

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

The incidence of non-melanocytic skin cancers in an Australian population: results of a five-year prospective study.

Two thousand, six hundred and sixty-nine persons who were aged 40 years and older attended for examination of the light-exposed areas of the head and neck, forearms and dorsa of the hands during a skin-cancer survey of the population of Maryborough, which was conducted annually for five years from 1982-1986 inclusive. Sixty (2.25%) persons--12 persons each with a squamous-cell carcinoma and 48 persons with a total of 51 basal-cell carcinomas--had at least one non-melanocytic skin cancer at the first examination. One thousand, nine hundred and eighty-one (74% of the study population) persons were seen on more than one occasion, which allowed for 6288 person-years of follow-up for the determination of the incidence of new cancers. The findings showed a calculated minimal age-standardized incidence rate of 873 non-melanocytic skin cancers/100,000 population each year. The minimal incidence rate for basal-cell carcinomas was 672 cases/100,000 population each year and for squamous-cell carcinomas was 201 cases/100,000 population each year. The rate ratio of the incidence of basal-cell carcinomas to that of squamous-cell carcinomas was 3.34 to one. Age, sex, skin reaction to sunlight and occupation all were significant factors in the determination of the risk of developing non-melanocytic skin cancers. The enormous costs that are involved in the treatment of non-melanocytic skin cancers and related lesions suggest that more time, effort and money need to be spent to reduce what has become a major public-health problem in Australia.

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.

The relationship of basal cell carcinomas and squamous cell carcinomas to solar keratoses.

Six thousand four hundred sixteen people aged 40 years and over from three different locations in Victoria (Australia) were examined on the hands, forearms, head, and neck for the presence of solar keratoses and basal (BCCs) and squamous cell carcinomas (SCCs). Analysis of the relationship between these tumors revealed that the factors which predicted the likelihood of developing a solar keratosis were essentially the same as those that predicted the likelihood of developing a BCC and/or an SCC. These were age, sex, years of residence in Australia, indoor or outdoor occupation, tanning ability, propensity to sunburn, and location of residence. The presence of a coexisting solar keratosis was necessary for the development of an SCC in contrast to the development of a BCC. The findings suggest that unlike BCCs, the majority of SCCs in light-exposed areas may arise from preexisting solar keratoses. Whereas the prevalence of BCCs and SCCs was relatively constant in the three locations, the prevalence of solar keratoses differed markedly in direct relation to the degree of isolation. This suggests that solar keratoses are a more sensitive indicator of sunlight exposure than invasive carcinoma.

Malignant transformation of solar keratoses to squamous cell carcinoma.

1689 people aged 40 years and over were examined over a 5-year period to determine the incidence of malignant transformation of solar keratoses. They were seen on 2 consecutive years on 4267 occasions; a total of 21,905 solar keratoses were present on the first visit. A squamous cell carcinoma (SCC) developed within 12 months on 28 of the 4267 occasions. Where accurate mapping of both SCCs and pre-existing solar keratoses was available, it was found that 10/17 (60%) SCCs arose from a lesion diagnosed clinically as a solar keratosis in the previous year and the other 7 (40%) SCCs on what had been clinically normal skin 12 months previously. The risk of malignant transformation of a solar keratosis to SCC within 1 year was less than 1/1000. The cost-effectiveness of treating all solar keratoses to prevent the development of SCC is questionable.

Spontaneous remission of solar keratoses: the case for conservative management.

One thousand and forty people aged 40 years and over, 616 (59.2%) of whom had solar keratoses, were followed for 12 months. Two hundred and twenty-four people (36.4%) had a spontaneous remission of at least one of their solar keratoses. A total of 485 lesions (25.9%) underwent spontaneous remission out of the 1873 lesions that were present at the first examination of these 224 people. There was no significant difference between the number of lesions present at the initial examination in those who had a spontaneous remission compared with those who did not. There was a 21.8% increase in the total number of solar keratoses in the 1040 people studied in the 12-month period, due to new lesions forming at the same time as remissions were occurring. The incidence rate of squamous cell carcinoma occurring in the people with solar keratoses was 0.24% for each solar keratosis present at the original examination. With a substantial proportion of solar keratoses remitting spontaneously, plus the low rate of malignant transformation and the low potential for metastasis to occur from squamous cell carcinoma arising in a solar keratosis, the rationale of treating all solar keratoses appears questionable.

Gynaecological and sexuality profile of new patients attending a family planning clinic.

PIP: 2067 first-time attenders (70% of all new patients during the 9-month study period) at a Family Planning Association clinic in Victoria, Australia, completed a questionnaire aimed at obtaining data on the age at onset of sexual activity and contraceptive use patterns. The mean age of study respondents was 23 years; only 13% were married. 86% of the women reported a history of sexual intercourse. The average age at 1st intercourse was 17.4 years, and over 50% were sexually active before 18 years of age. 51% did not use contraception at the time of 1st intercourse, and this rate was lower (37%) among women aged 15 years or less at coitarche. Of those who had used contraception, the most common methods were condoms (52%) and oral contraceptives (37%). When asked to rate the quality of their 1st act of sexual intercourse, 12% indicated the experience was better than they expected, 50% said it was as good as expected, and 38% indicated it was worse than anticipated. 90% claimed that the quality of their sexual experiences has improved since the 1st time. In terms of current frequency of sexual intercourse, 60% reported sexual relations less than twice a week, 26% have sex 2-3 times a week, and 14% have intercourse more than 3 times a week. When asked about their recent contraceptive practice, 47% reported contraception is always used, 26% use contraception most of the time, 10% use contraception sometimes, 5% rarely use such methods, and 12% never use family planning. Since these subjects were either already sexually active or were planning to initiate sexual activity, they are not representative of Australia's female population as a whole. However, they are an important target for family planning education programs.^ieng

Solar keratoses. The association with erythemal ultraviolet radiation in Australia.

The prevalence rate of solar keratoses among 2000 residents of Melbourne, Australia, was compared to the rate among 2113 residents of Maryborough, a north central Victorian city. There was a significantly higher prevalence rate among the Australian-born population of Maryborough compared with Melbourne residents of the same age, sex, country of birth, and level of outdoor activity. Calculation of the erythemal ultraviolet radiation level revealed a 14.2% increase in the dose in Maryborough compared with that in Melbourne. These figures demonstrate a significant increase in the rate of solar keratoses, and thus the potential for the development of skin cancer, in all of the age groups studied. The difference was associated with a relatively small increase in ultraviolet radiation between two areas that are separated by a latitude distance of only 110 km.

Non-melanotic skin cancer and solar keratoses in Victoria.

The light-exposed areas of the head and neck, forearms, and dorsum of each hand were examined in 2113 adults during a one-week study. The study was conducted in the isolated Victorian rural city of Maryborough which is situated in the northern part of the Central Highlands district. Of 2113 subjects, 49 (2.32%) had at least one skin cancer and 1202 (56.9%) had at least one solar keratosis. Histological confirmation of all skin cancers and doubtful solar keratoses was obtained. Age, skin type, and sunlight exposure were the major factors influencing the prevalence of solar keratoses and skin cancers.

Acupuncture treatment of chronic back pain. A double-blind placebo-controlled trial.

Acupuncture treatment of chronic low back pain was studied in a placebo-controlled double-blind crossover trial completed by 77 patients. The patients had significantly increased depression, neuroticism, and hypochondriasis scores. Initial pain levels correlated with state-anxiety, depression, pain duration, and abnormal illness behavior measures, as well as with the intake of psychotropic but not analgesic medication. Overall reduction in pain score was 26 percent for acupuncture and 22 percent for placebo treatment; the difference was not significant (p greater than 0.6). Analgesic drug intake was reduced to a similar extent in both groups. During the first phase of treatment, patients receiving acupuncture had a greater but not significantly different reduction in pain rating scores compared with those receiving placebo (t = 0.52; p greater than 0.6). This group showed significantly lower pain scores (p less than 0.05) in the second phase of the trial while receiving placebo treatment. Overall reduction in individual patient's pain score was best predicted by initial pain severity (r = 0.43; p less than 0.001) and psychotropic drug intake (r = 0.37; p less than 0.001). None of the variables tested predicted which patients would specifically respond to acupuncture or placebo.