Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides.

The gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.

Protein tyrosine phosphorylation in signalling pathways leading to the activation of gelatinase A: activation of gelatinase A by treatment with the protein tyrosine phosphatase inhibitor sodium orthovanadate.

Fibroblasts in monolayer culture secrete gelatinase A (MMP2; 72 kDa type IV collagenase) only in its proenzyme form. Unlike other secreted matrix metalloproteinases, progelatinase A is refractory to activation by serine proteinases. Disparate agents, including monensin, cytochalasin D, and concanavalin A, have been found to mediate the activation of gelatinase A zymogen secreted by fibroblast monolayers. Our finding that monensin-mediated activation can be reversed by the protein tyrosine kinase inhibitor genistein (Li et al., Experimental Cell Research 232 (1997) 332) prompted us to investigate the effect of the specific inhibitor of protein tyrosine phosphatases, sodium orthovanadate, on progelatinase A activation. Treatment of fibroblast monolayers with orthovanadate also results in the secretion of activated gelatinase A. This activation is dose- and time-dependent, requires protein synthesis, and is associated with cell membranes. Vanadate-mediated activation does not occur in the presence of herbimycin A, a protein tyrosine kinase inhibitor. As with progelatinase activation mediated by monensin, concanavalin A, and cytochalasin D, orthovanadate treatment results in increased synthesis of the membrane proteinase MT1-MMP, that can catalyze the activation of progelatinase A. Protein tyrosine kinase inhibitors are able to prevent the increase of MT1-MMP mRNA, as shown by Northern blot and RT-PCR. In addition, orthovanadate potentiates the effects of monensin and concanavalin A. While treatment with monensin or concanavalin A result only in an increase of the putative activator MT1-MMP, orthovanadate also reduces the production of the specific inhibitor TIMP-2. These experiments implicate protein tyrosine phosphorylation in the signal transduction pathways which lead to the activation of progelatinase A.

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

Regulation of gelatinase in human skin organ cultures by glucocorticoids.

Hydrocortisone and dexamethasone prevent the appearance of gelatinase in serum-free explant cultures of normal human skin. Hydrocortisone inhibits maximally at 10(-6) M and dexamethasone at 10(-8) M in culture medium. Glucocorticoids at these concentrations do not cause a generalized decrease in protein synthesis; thus the effect on gelatinase shows specificity. The reduction in gelatinase activity caused by dexamethasone can be overcome in the presence of dexamethasone 21-mesylate, a glucocorticoid antagonist that binds irreversibly to the cytoplasmic steroid receptor. These data suggest that the enzymes of collagen degradation, collagenase and gelatinase, may be coregulated.

Retinoic acid inhibition of collagenase and gelatinase expression in human skin fibroblast cultures. Evidence for a dual mechanism.

Human skin fibroblast cultures have been employed to study the effects of a variety of vitamin A analogues (retinoids) on the expression of two enzymes involved in collagen degradation in the skin, collagenase and a gelatinolytic protease. In normal and recessive dystrophic epidermolysis bullosa fibroblast cultures, retinoic acid compounds were effective inhibitors of the accumulation of both enzymes in the culture medium with half-maximal inhibitions occurring at 0.25-1 microM for collagenase and at 3-6 microM for the gelatinolytic protease. Various retinoids exhibited differing degrees of inhibitory actions, so that at a 1 microM concentration, relative inhibitions were: 13-cis-retinoic acid greater than all-trans-retinoic acid greater than aromatic retinoid (Ro 10-9359) much greater than retinol. The retinoic acid-mediated decrease in collagenase activity was accompanied by a parallel decrease in immunoreactive collagenase protein, suggesting that the retinoic acids were acting to inhibit synthesis of the enzyme. However, an additional effect of these agents was encountered. Although the retinoids themselves had no direct collagenase inhibitory action, medium derived from cultures maintained in these retinoids showed direct inhibitory capacity which was dependent both on the concentration of retinoic acid and on the length of time in culture. The results suggest that the retinoic acids modulate collagenase in vitro by two mechanisms: by decreasing the synthesis of enzyme protein and by modulating the expression of an inhibitory molecule.

Generalized dominant epidermolysis bullosa simplex: decreased activity of a gelatinolytic protease in cultured fibroblasts as a phenotypic marker.

To characterize biochemical traits associated with various forms of epidermolysis bullosa (EB), we used skin fibroblast cultures to measure a gelatin-specific neutral metalloprotease. Compared to normal cultures, levels of this gelatinase were 7-fold decreased in cell cultures from 3 patients from 3 kindreds with generalized dominant EB simplex of the Koebner type (DEBS-K) (p less than 0.001). The specificity of this trait was shown in several ways. The growth kinetics and total protein synthesis of the cells were unaltered. The activity of lactic dehydrogenase, a cytoplasmic enzyme, was also unaltered, indicating the integrity of the cells was not compromised. The decrease in gelatinase activity is specific for generalized DEBS-K, since cultures from recessive dystrophic EB, recessive junctional EB, dominant dystrophic EB, and a second genetic type of DEBS all fail to show this defect. However, since it has been suggested that DEBS-K and the localized form of DEBS, the Weber-Cockayne type (DEBS-WC), may represent allelic mutations of varying severity, we measured gelatinase activity in cell cultures of 13 patients of this type. The levels displayed a biphasic segregation in which 7 of 13 values were equal to, or greater than, the mean activity of control cells. Cultures from the remaining 6 patients were greater than 1 SD below the mean control value and approximated those seen in the generalized DEBS-K patients. These studies suggest that the decrease in gelatinolytic activity is a marker for DEBS-K and that DEBS-K and DEBS-WC may be closely related genetic disorders in which the defect in gelatinolytic protease represents a pleiotropic effect of the gene for DEBS or is genetically linked to the DEBS gene.

Fluorometric determination of pyruvate and alpha-ketoglutarate in cerebrospinal fluid and plasma of infants and children. A simple test that screen for metabolic disorders.

Levels of pyruvate and alpha-ketoglutarate in the cerebrospinal fluid (CSF) of 26 children, aged 4 months to 5 1/2 years, with febrile seizures and of 19 children, aged 4 months to 14 years, with the diagnosis of epilepsy were not different from values seen in 119 "normal" children 8 days to 14 years of age. The CSF samples from 24 adults, 24 to 81 years of age, suspected of having a herniated disk were also examined. In the pediatric age group, the data showed a highly significant downward trend of CSF and plasma alpha-ketoglutarate values with age; pyruvate values did not change. A correlation of the values of the two keto acids in the blood and CSF of 42 other children without apparent neurologic disease was also made. Findings in a child with thiamine deficiency suggest that CSF alpha-ketoglutarate may be a more sensitive indicator of deficiency than plasma alpha-ketoglutarate or pyruvate. Measurements of these keto acids in plasma and CSF may be diagnostically useful in a variety of metabolic disorders. Findings in 155 children from birth (20 minutes) to 17 years of age without neurologic disease are submitted as a standard of reference.

A dose-response evaluation of pipecuronium bromide in elderly patients under balanced anesthesia.

Pipecuronium bromide is a new steroidal non-depolarizing muscle relaxant currently under investigation. It is similar to pancuronium with respect to the duration of action, but lacking its cardiovascular side effects. We examined the dose-response relation of pipecuronium in 27 patients, ages 66-79 years, utilizing the incremental dose method under balanced anesthesia. The ED50, ED90 and ED95 were 22.42 (5.2) mcg/kg, 31.81 (6.9) mcg/kg and 35.12 (7.8) mcg/kg, respectively (log probit method). Our recovery data also demonstrate that residual neuromuscular blockade due to pipecuronium can easily be antagonized with neostigmine as long as spontaneous recovery of T1- at the time of reversal administration is greater than 13%. The authors conclude that under balanced anesthesia the cumulative dose-response of pipecuronium in the elderly patients is consistent with those previously described for younger population. Therefore, no dose adjustment appears necessary for the elderly. However, as with all medications, careful administration is appropriate.

Pain control for the urologist.

Pain states can be divided into three categories: acute, chronic not resulting from malignancy, and chronic malignant pain. A Pain Clinic can provide in-depth evaluation and treatment of difficult pain problems. A variety of oral and parenteral medications, ranging from nonsteroidal analgesics to narcotics, are available to control pain. Local anesthetics can be used for local infiltration, and peripheral and central nerve blocks can also be used as indications warrant.

Does the use of methylmethacrylate cement in total shoulder replacement induce hemodynamic or pulmonary instability?

STUDY OBJECTIVE: To investigate whether the use of methylmethacrylate cement causes hemodynamic or pulmonary instability during total shoulder replacement surgery. DESIGN: Prospective, nonrandomized study. SETTING: Operating room. PATIENTS: 9 ASA physical status I and II patients. INTERVENTIONS: A 20-gauge radial artery catheter was placed in the wrist opposite the surgical site. Sedation with midazolam was provided, and a pulmonary artery catheter was placed through an 8.5-Fr introducer into the patient's right internal jugular vein. MEASUREMENTS AND MAIN RESULTS: Before induction of anesthesia, systolic, diastolic, and mean arterial blood pressures; heart rate; central venous pressure; systolic, diastolic, and mean pulmonary artery pressures; pulmonary capillary wedge pressure; and thermodilution cardiac output measurements were obtained. Arterial and mixed venous blood gas samples also were collected and analyzed for calculation of Qs/Qt. These hemodynamic and pulmonary parameters were measured again just before cementing of each prosthesis with methylmethacrylate cement and at 1, 5, 10, and 20 minutes after cementing. There were no statistically significant changes in any of the measured hemodynamic parameters at any time. There was no statistically significant difference in the calculated intrapulmonary shunt fraction. CONCLUSION: In this study population, the use of methylmethacrylate for total shoulder replacement was not associated with adverse hemodynamic events or increased intrapulmonary shunting.

A risk-specific anesthesia consent form may hinder the informed consent process.

STUDY OBJECTIVE: To evaluate the effect of a preprinted, risk-specific consent form on the amount of anesthetic risk information patients retain from the preoperative interview. DESIGN: Postoperative survey of consecutive inpatients to determine risk information retained before and after implementation of a preprinted anesthesia consent form, using standard preoperative risk discussions. SETTING: Inpatient units of a university medical center. PATIENTS: Two groups of patients, both of whom received a standard oral discussion of anesthetic risk information, were compared. Patients in the control group (125 consecutive inpatients) received this information only orally and were interviewed two weeks prior to implementation of a preprinted anesthesia consent form. Patients in the study group (92 consecutive inpatients) received this information orally and via a preprinted consent form and were interviewed between the fourth and sixth weeks after implementation of a preprinted anesthesia consent form. INTERVENTIONS: Anesthesia residents discussed five standard anesthetic risks with elective, adult inpatients (n = 233) during a two-week period immediately before and between the fourth and sixth weeks after instituting the mandatory use of a risk-specific anesthesia consent form. These patients were interviewed postoperatively by one of the authors to determine the amount of anesthesia risk information they retained. MEASUREMENTS AND MAIN RESULTS: Results of the postoperative survey showed that patients in the control group retained more information concerning anesthetic risks than did those in the study group (33% vs 19%, p less than 0.01). CONCLUSIONS: To improve the informed consent process, either a better method of presenting the preprinted, risk-specific consent form or another method of simultaneously conveying and documenting risk information is needed.

Medical waste in the environment: do anesthesia personnel have a role to play?

STUDY OBJECTIVE: To conduct a feasibility study of the mechanics of recycling single-use anesthesia breathing systems and practices of anesthesiologists and nurse-anesthetists in a tri-state region. STUDY DESIGN: Two-part, open, prospective analysis using pre-printed questionnaire and cost/time analysis of labor and materials. SETTING: Questionnaire sent to 413 anesthesiology departments in Pennsylvania, New Jersey, and Delaware, and hospital/recycling facility for evaluation of time and cost. MEASUREMENTS AND MAIN RESULTS: Time to disassemble and sort the breathing circuits, analysis of costs and obtainable income from byproducts of recycling, and standard survey questionnaire concerning demographic characteristics of respondents and individual department/hospital practitioners. Data analysis included analysis of variance and Kruskal-Wallis tests. Pilot analysis: Sorting of circuits to economic component required ten minutes at an average cost of $1.60 Value of scraps obtainable was $3.44, leaving a gross margin of $1.84 for a box of 18 circuits. Benefit analysis: Extended reduction in the regulated medical waste in our operating room of 16,875 lb, saving $4,387.50 per year. With generation of revenue from scrap, the net gain is $5,994.64 per yr. Questionnaire: Majority (83%) of departments polled would participate in recycling implemented by suppliers. Most respondents would not consider (58%) recycling unless mandated by law. CONCLUSION: The program described is cost-effective and environmentally beneficial.

A comparison of desflurane and isoflurane in prolonged surgery.

STUDY OBJECTIVE: To compare desflurane with isoflurane in several anesthetic situations. DESIGN: Intubating conditions, hemodynamic response to intubation, maintenance hemodynamics, and speed of recovery from desflurane and isoflurane anesthesia were evaluated. In addition, interaction with a muscle relaxant at low and high concentrations of the anesthetics were compared. SETTING: Thomas Jefferson University Hospital. PATIENTS: Thirty-two patients who received general anesthesia for lengthy, mostly orthopedic procedures. INTERVENTIONS: Immediately after induction with thiopental sodium, desflurane or isoflurane in nitrous oxide-oxygen was administered via face mask. Anesthesia was deepened until end-tidal concentration reached 1.7 minimum alveolar concentration (MAC). The trachea was intubated without the aid of a muscle relaxant. Heart rate (HR) and blood pressure (BP) were recorded before and at 1, 2, 4, 5, and 10 minutes after intubation. Noninvasive cardiac output (CO) and systemic vascular resistance (SVR) were determined while the patient was awake, immediately before intubation, and at 5 and 10 minutes after intubation. Following intubation, the concentration of desflurane or isoflurane was lowered until the end-tidal concentration reached 0.65 MAC (low-MAC group), 1.25 MAC (high-MAC group), or 0 MAC (control group). Pancuronium bromide in 0.005 mg/kg doses was administered incrementally until T1 (first twitch of train-of-four) was depressed more than 90%. ED50 and ED95 for pancuronium with balanced anesthesia and for desflurane or isoflurane in low and high MACs, as well as speed of recovery, were determined. The time to responsiveness and awakening also was determined. MEASUREMENTS AND MAIN RESULTS: There was no significant difference between desflurane and isoflurane in intubating conditions or in BP or HR response to tracheal intubation. Both anesthetics increased HR significantly during induction. BP rose with desflurane at the preintubation point; other points showed no difference. A hyperdynamic response of increased HR and BP above 20% of baseline values was seen more frequently with desflurane (n = 7) than with isoflurane (n = 1). CO was elevated at all times after induction for low and high concentrations of both drugs, while SVR decreased over the same time with no significant difference between drugs. ED50 and ED95 for pancuronium were similar under desflurane and isoflurane at both low and high MAC, but they were significantly lower than under balanced anesthesia. Awakening times were similar for desflurane and isoflurane. CONCLUSIONS: Desflurane is similar to isoflurane in providing anesthesia for intubation and maintenance. Desflurane tends to increase HR and occasionally causes a hyperdynamic response during rapid deepening of anesthesia. It is very similar to isoflurane in its interaction with pancuronium.

The effect of the prone position on venous pressure and blood loss during lumbar laminectomy.

STUDY OBJECTIVE: To determine the effects of three different prone support systems (Andrews spinal surgery frame, Cloward surgical saddle, and longitudinal bolsters) on inferior vena cava (IVC) and superior vena cava (SVC) pressures; the validity of measuring central venous pressure (CVP) for the determination of ideal positioning of the patient; and the relationship among frame type, blood loss, and hemodynamic measurements. DESIGN: Prospective, randomized study of the hemodynamic effects of the prone position. SETTING: Inpatient surgery at a university hospital (regional spinal cord injury treatment center). PATIENTS: Eighteen patients free of significant coexisting disease (ASA physical status I and II) undergoing elective lumbar laminectomy. INTERVENTIONS: Patients were assigned to one of three support frames and measurement of SVC pressure, IVC pressure, and mean arterial pressures (MAP) were obtained supine, prone, and after repositioning. These pressures and measured blood loss were obtained every 15 minutes during the surgical laminectomy portion of the procedure. MEASUREMENTS AND MAIN RESULTS: Patients positioned on the Andrews frame had decreased mean SVC and IVC pressures from 8.7 mmHg and 8.4 mmHg in the supine position to 3.3 mmHg and 1.8 mmHg in the prone position, respectively (p less than 0.001). Prone position CVP also was significantly lower in the Andrews group compared with that in the other two groups (p less than 0.001). Repositioning efforts did not significantly decrease CVP. Blood loss was higher in the Cloward group (1,150 +/- 989 ml) than in the Andrews (245 +/- 283 ml) and bolsters (262 +/- 188 ml) groups (p less than 0.02). CONCLUSIONS: Increased blood loss was not associated with increased SVC or IVC pressure, nor was there any significant correlation between any demographic or hemodynamic variable and blood loss. There was no evidence that CVP is useful in determining the ideal prone position in patients undergoing lumbar laminectomy.

Intubating conditions after pipecuronium bromide: the influence of dose and time.

STUDY OBJECTIVE: To determine the intubating conditions following the administration of pipecuronium bromide in doses of two (0.07 mg/kg) or three (0.1 mg/kg) times ED95 (average dose that gives 95% block of the first twitch). DESIGN: To compare intubating conditions at 11/2 and 21/2 minutes in 41 patients receiving balanced anesthesia. SETTING: Surgical patients at Thomas Jefferson University Hospital. PATIENTS: Forty-one patients undergoing surgical procedure who received general anesthesia. INTERVENTIONS: After obtaining a stable baseline of train-of-four (TOF), 41 patients randomly received either 0.07 mg/kg or 0.1 mg/kg of pipecuronium as a single intravenous (IV) bolus dose, and the trachea was intubated either at 11/2 or 21/2 minutes. MEASUREMENTS AND MAIN RESULTS: Intubating conditions at 21/2 minutes appeared significantly better than those at 11/2 minutes, regardless of the pipecuronium dose. The mean time for T1 (first twitch of TOF) to reach 50% and 90% suppression was 1.36 +/- 0.51 minutes and 2.29 +/- 0.8 minutes, respectively, for the 0.07 mg/kg dose and 1.07 +/- 0.27 minutes and 1.72 +/- 0.45 minutes, respectively, for the 0.1 mg/kg dose. This did not make a significant difference in intubating conditions at either time. The time to 25% recovery of T1 was 68.2 +/- 22 minutes for the 0.07 mg/kg dose and 121.5 +/- 49 minutes for the 0.1 mg/kg dose. In patients who had spontaneous recovery of T1 to between 10% and 25% of control, administration of neostigmine or edrophonium resulted in identical recovery in 10 minutes. However, in patients with less than 10% spontaneous recovery of T1, neostigmine appeared to be superior to edrophonium. CONCLUSION: Pipecuronium has a relatively rapid onset. The trachea could be intubated successfully in 11/2 minutes with a dose of either 0.07 mg/kg or 0.1 mg/kg. If the clinical situation requires perfect relaxation with no movement or bucking, we recommend waiting at least 21/2 minutes.

Decrease in arterial pressure following heparin injection prior to cardiopulmonary bypass.

Evidence exists in the literature that heparin has vasodilating properties. We recorded arterial blood pressure changes that occur after administration of 300 units/kg of beef-lung heparin prior to cardiopulmonary bypass. In 37 out of 43 patients there was a significant decrease in arterial pressure following administration of beef-lung sodium heparin. Systolic blood pressure fell approximately 10 mmHg (1.3 kPa) and diastolic blood pressure fell about 5 mmHg (0.65 kPa). Heart rates remained unchanged. In 7 of the 43 patients in whom pulmonary artery catheters were placed, cardiac output was determined and systemic vascular resistance was calculated. On the basis of these calculations, it seems that a decrease in systemic vascular resistance is responsible for the decrease in arterial blood pressure noted following sodium heparin.