Therapeutic intervention scoring system (TISS)--a method for measuring workload and calculating costs in the ICU.

The Therapeutic Intervention Scoring System (TISS) is an easily applicable method for measurement of workload in the intensive care unit (ICU). In the present study a modified TISS-scoring (mTISS) was performed daily during 1988-1989 on 2693 patients in a general ICU. Of these, 900 could be classified as 'true' ICU-patients (ICU-stay > or = 24 h or TISS > or = 20 points), whereas the rest were postoperative. In ICU-patients the average length of stay was 4.5 +/- 8.9 days and the average workload 114 +/- 218 mTISS-points. The workload was not significantly related to age or type of admission (scheduled vs unscheduled). Hospital non-survivors (13.6%) showed a significantly increased mean total mTISS-score (239 +/- 364, P < 0.001). Critically ill (TISS Class IV) patients (14% of the sample), with an average workload of 437 +/- 401 mTISS-points, consumed 53% of the total resources. Patients categorized (ICD-9) to respiratory and infectious diseases showed the greatest average workload (207 +/- 315 and 208 +/- 355 mTISS-points, respectively). A workload-index was also developed relating the actual workload to the ICU personnel. The cost of each mTISS-point was calculated. In conclusion, the present study showed that mTISS is a valuable tool when evaluating resource utilization in the ICU. Together with the proposed workload-index and calculation of costs, mTISS could be used for ICU management control.


Sublingual premedication with brotizolam.

This randomized, double-blind and double-dummy study was carried out in order to compare the perioperative sedation after premedication with either brotizolam 0.25-0.50 mg sublingually or diazepam 5-10 mg orally. Sixty-two patients aged 18-60 years scheduled for minor gynaecological surgery in general anaesthesia were included. Assessments were: 1. auditory continued response time (ACRT); 2. coma scale; 3. anxiety scale; and 4. final patient questionnaire. One hour after premedication the brotizolam group was more sedated, based on ACRT (P < 0.01) and the coma scale (P < 0.05). The final questionnaire showed (P < 0.05) that the brotizolam group was more satisfied with the effect of the premedication. Seven hours after the premedication the ACRT scores in both groups were similar to those before premedication and all the patients could walk about freely. In conclusion, as a premedicant in outpatients sublingual brotizolam appears to be a good alternative to diazepam.


Recollections of general anaesthesia: a survey of anaesthesiological practice.

In order to identify the factors to which patients attach importance when undergoing general anaesthesia, 678 patients were retrospectively asked about their recollections of previous anaesthetics during routine preoperative screening over a period of 14 months. The most frequently mentioned recollections concerned the post-anaesthetic period, followed by recollections of the induction of anaesthesia. From the post-anaesthetic period, nausea/vomiting and drowsiness were most often cited. The number of anaesthetics previously undergone had no influence on the reports of the two most frequently mentioned complaints. The rate of nausea/vomiting in this series was 21.0%. The number of patients reporting nausea/vomiting following an anaesthetic has not changed over the years. The last 250 patients were asked to assess the quality of their anaesthetics on a 5-point scale. More than a quarter of the patients were not satisfied with the anaesthesia. No correlation was found between the assessment of the anaesthesia and complaints about nausea/vomiting and drowsiness. The possible role of psychological factors in the origin of complaints about anaesthesia is discussed.


Lidocaine pretreatment effectively decreases the incidence of hiccups during methohexitone administration for dilatation and curettage.

The efficacy of lidocaine 1 mg.kg-1 for prevention of methohexitone-induced hiccups was assessed in a double-blind fashion in 200 patients undergoing dilatation and curettage for pregnancy interruption. The patients were randomly assigned to receive either lidocaine 1% or saline 0.9% in a similar way (1 ml for every 10 kg of body weight). The incidence of hiccups was 16% in the control group compared to 6% in the lidocaine group. We speculate that this reduction in the methohexitone-induced hiccup ratio is related to lidocaine's membrane-stabilizing properties, which decrease the excitability of all the nervous structures involved in this reflex.


A device for measuring the lateral wall cuff pressure of endotracheal tubes.

A new method for measuring the lateral wall pressure on the trachea from cuffs of endotracheal tubes is presented. The method is based on measuring the force necessary to force a small, constant flow of air through a Teflon or silicone rubber envelope, placed between the cuff and the tracheal wall. The pressures recorded are 0.1 to 0.3 kPa higher than the theoretically correct values. The response time is 0.28 s for 90% of full deflection. Dynamic recordings of the lateral wall pressure of high and low residual volume cuffs can be obtained for analysis of the interaction between the cuff and the tracheal compliance. No method for accurate, dynamic recording of the lateral wall cuff pressure has previously been published.


Clinical and metabolic responses to different kinds of premedication in ASA III patients.

Clinical and metabolic responses to atropine plus pethidine and to scopolamine plus morphine premedication were studied in 45 ASA physical status III patients undergoing gynaecological procedures. Atropine 0.5 mg plus pethidine 50 mg intramuscularly (Group 1), scopolamine 0.24 mg plus morphine 8 mg (Group 2), or intramuscular placebo (Group 3) premedication were given in random, double-blind fashion. Scopolamine-morphine premedication caused a significant decrease in energy expenditure (EE) and oxygen consumption (VO2) (from 1229 +/- 193 to 1184 +/- 221 kcal/24 h, P = 0.004 and from 105 +/- 11 to 102 +/- 12 ml/min/m2, P = 0.006, respectively) simultaneously with a decrease in rate-pressure product (RPP) (P = 0.0001) and an increase in pressure-rate quotient (PRQ) (P = 0.034). Atropine-pethidine premedication induced a decrease in RPP but not in EE or VO2. In the placebo group both RPP and VO2 first increased and then slowly returned to the levels measured prior to premedication. The RPP was significantly lower in Group 2 than in Groups 1 and 3 at both 30 and 60 min. The degrees of subjective tiredness and anxiolysis were significantly greater in Groups 1 and 2 (showing good sedative and anxiolytic effect) than in Group 3. These results show that in ASA III patients, atropine-pethidine premedication does not decrease the sympathoadrenal reaction to the degree its anxiolytic and sedative effect would suggest. This may indicate neuroendocrine stress induced by atropine-pethidine.


The effects of propofol, methohexitone and isoflurane on the baroreceptor reflex in the cat.

The effects of propofol (P), methohexitone (M) and isoflurane (I) on the baroreceptor reflex were studied in a cat model in which the blood pressure in a bilateral isolated carotid sinus preparation was artificially varied between 50-200 mmHg. The influence from aortic and cardiopulmonary baroreceptors was excluded by vagotomy. With basal chloralose anaesthesia as control, the investigated anaesthetics were used in doses corresponding to MAC 0.5 and 1.0. The maximum change in systemic mean arterial pressure (MAP) and heart rate (HR) following a defined increase in carotid sinus pressure was used as an index of baroreceptor reflex sensitivity. Compared to control, M and I anaesthesia were associated with significant depression of baroreceptor reflex sensitivity at the high dose (corresponding to MAC 1.0), and during I anaesthesia also at the low dose (MAC 0.5). The baroreceptor reflex sensitivity was maintained during propofol anaesthesia. The carotid sinus pressure interval at which the maximum changes in MAP could be elicited, was significantly higher during M than during P. This indicates resetting of the baroreflex.


Ventilatory effects of epidural clonidine during the first 3 hours after caesarean section.

Many authors have shown the analgesic efficacy of 150-800 micrograms of epidural clonidine in the postoperative period. Its use as an analgesic after caesarean section has recently been studied with higher dosages (400-800 micrograms). Our study aimed at assessing the analgesic and ventilatory effects of two smaller doses of epidural clonidine (150 and 300 micrograms), which were compared to the effects of 10 mg of parenteral morphine (M) during the first 3 h after caesarean section. The duration of the analgesic effect was longest with 150 micrograms of epidural clonidine. Arterial blood pressure decreased from 30 min after the injection to the end of the study in both epidural clonidine groups. A marked sedation was observed in patients receiving 300 micrograms of epidural clonidine and was frequently associated with snoring, obstructive apnoea and episodes of arterial oxygen desaturation. We conclude that 150 micrograms of epidural clonidine provides better and longer analgesia after caesarean section than 10 mg of parenteral morphine, and seems preferable to higher doses (300 micrograms) in this setting, since 300 micrograms of epidural clonidine may produce unacceptable respiratory obstructive disturbances.


Elimination of apparatus dead space--a simple method for improving CO2 removal without increasing airway pressure.

During mechanical ventilation the apparatus dead space can be eliminated by insufflating through one lumen of a tracheal double-lumen tube and allowing expiration through the other. In six intravenously anesthetized pigs, this technique resulted in an 18% (1 kPa) decrease in PaCO2 compared with insufflating through both lumens (32 ml rebreathing volume). Oxygenation, airway pressures, and tidal volumes were unchanged. Flushing the trachea with fresh gas during the expiratory phase did not improve the efficiency of ventilation. It is concluded that elimination of apparatus dead space improves CO2 removal without increasing airway pressures and tidal volumes, and it is suggested that minimization of apparatus dead space should be tried before more advanced ventilatory modes are considered.


Gas exchange impairment and pulmonary densities after cardiac surgery.

In 11 patients with impaired respiratory function after coronary artery revascularization surgery, thoracic computed tomography (TCT) and cardiopulmonary profile were obtained. The patients were haemodynamically stable without clinical or radiological signs of lung oedema. Oxygenation was reduced in all patients (alveolo-arterial PO2-difference (PA-aO2) = 37.3 +/- 10.39 kPa, venous admixture (QVA/QT) = 26.4 +/- 4.4%) during mechanical ventilation with positive end-expiratory pressure (PEEP = 5 cmH2O) (0.5 kPa). TCT-scan analysis revealed considerable amounts of crest-shaped bilateral densities in dependent lung regions. There were no differences between the right and left hemithorax. Atelectatic lung tissues were defined as areas presenting with attenuation values of -200 to +20 Hounsfield Units. The magnitude of non-ventilated areas correlated with QVA/QT (r = 0.875, P < or = 0.01), but not with the duration of either extracorporeal circulation, surgical procedure or general anaesthesia. It is concluded that atelectasis in dependent lung areas contributes to impaired gas exchange after cardiac surgery.


Hemodynamic interactions when combining verapamil, acute changes in extracellular ionized calcium concentration and enflurane, halothane or isoflurane in chronically instrumented dogs.

To assess the hemodynamic interactions when combining verapamil, acute changes in extracellular ionized calcium concentration [Ca2+] and enflurane (2.5%), halothane (1.2%) or isoflurane (1.6%), seven dogs were chronically instrumented to measure heart rate (HR), aortic, left atrial and left ventricular (LV) pressures, and cardiac output (CO). [Ca2+] was lowered 0.35 mmol.l-1 by citrate infusion and then increased 0.35 mmol.l-1 above control level by CaCl2 infusions. Verapamil was infused at 3 micrograms.kg-1 x min-1 (loading dose 200 (awake), 150 (isoflurane) or 100 (enflurane and halothane) micrograms.kg-1), giving mean verapamil concentrations around 75 (range of means: 66-84 ng.ml-1). Verapamil produced mostly minor changes in the cardiovascular effects of changing [Ca2+] in both awake and anesthetized dogs, indicating mostly additive effects. Verapamil induced a decrease in HR at high [Ca2+] and abolished an increase in mean aortic pressure at both low and high [Ca2+] awake. Verapamil exaggerated the decrease in CO and stroke volume (SV) induced by low [Ca2+] during enflurane anesthesia and abolished the increase in CO induced by low [Ca2+] and exaggerated the increase in SV and LV dP/dtmax induced by high [Ca2+] during halothane anesthesia.


Anaesthetic practice and postoperative pulmonary complications.

The aim of this study was to identify risk factors associated with postoperative pulmonary complications. The influence of the anaesthetic technique was evaluated (i.e. general contra regional anaesthesia and long contra intermediately acting muscle relaxants (pancuronium and atracurium)) taking into account the patient's age, the presence or absence of chronic obstructive lung disease (preoperative risk factors), the type of surgery and the duration of anaesthesia (perioperative risk factors). Seven thousand and twenty-nine patients undergoing abdominal, urological, gynaecological or orthopaedic surgery were included in the study. A total of 290 patients (4.1%) suffered from one or more postoperative pulmonary complications. Six thousand and sixty-two patients received general anaesthesia and 4.5% of these had postoperative pulmonary complications. Of the patients admitted to major surgery receiving pancuronium, 12.7% (135/1062) developed postoperative pulmonary complications, compared to only 5.1% (23/449) receiving atracurium (P < 0.05). When stratified for type of surgery and duration of anaesthesia, conventional statistics showed no difference between pancuronium and atracurium as regard postoperative pulmonary complications. However, a logistic regression analysis indicated that long-lasting procedures involving pancuronium entailed a higher risk of postoperative pulmonary complications than did other procedures. In patients having regional anaesthesia, only 1.9% (18/967) developed postoperative pulmonary complications (P < 0.05 compared to general anaesthesia). However, when stratified for type of surgery there was a significantly higher incidence of postoperative pulmonary complications only in patients undergoing major orthopaedic surgery under general anaesthesia, 11.5% compared to 3.6% in patients given a regional anaesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)


Core temperature measured in the auricular canal: comparison between four different tympanic thermometers.

Four different tympanic thermometers, absorbing infra-red radiation, (FirstTemp, Diatek, Ivac and Genius) were studied. Variations from repeated measurements and reliability of tympanic temperature compared to oesophageal, rectal and pulmonary artery temperature were studied. Core temperature measured by the "FirstTemp" and the "Genius" thermometers showed slightly higher values than core temperature measured by oesophageal, rectal and pulmonary artery thermistors. Compared to oesophageal temperature "FirstTemp" showed 0.56 degrees C and "Genius" 0.28 degrees C higher values, while the "Diatek" and the "Ivac" gave slightly lower values (-0.5 degrees C and -0.28 degrees C). All four thermometers were found accurate for repeated measurement both in terms of standard deviation and coefficient of variance. All four tympanic thermometers showed good accuracy for changes in core temperatures compared to the reference thermistors (r2 values 0.96 for "FirstTemp", 0.88 for "Diatek", 0.96 for "IVAC" and 0.95 for "Genius"). The tympanic thermometer was found to be a valuable alternative for measuring core temperature in most clinical settings.


The effect of acute hypothermia and serum potassium concentration on potassium cardiotoxicity in anesthetized rats.

We examined the effects of hypothermia on serum K+ concentration and the interaction of body temperature and K+ load on cardiac toxicity in anesthetized rats. Serum K+ concentration significantly decreased to 2.61 +/- 0.13, 2.59 +/- 0.19 and 2.39 +/- 0.14 mmol/l at 31.0 degrees C, 28.0 degrees C and 25.0 degrees C, respectively, from the control value of 2.80 +/- 0.15 mmol/l at 37.0 degrees C. We used a 300% increase in baseline QRS duration as evidence of cardiac toxicity. Serum K+ concentrations of 4.95 +/- 0.12, 4.71 +/- 0.10, 4.45 +/- 0.14 and 4.07 +/- 0.14 mmol/l resulted in cardiac toxicity at 37.0 degrees C, 31.0 degrees C, 28.0 degrees C, and 25.0 degrees C, respectively. These data indicate that the level at which an elevation of serum K+ concentration causes cardiac toxicity diminishes with progressive hypothermia. We conclude that hypothermia induces hypokalemia, possibly through redistribution, and that the myocardium appears to be more sensitive to the toxic effects of K+ as hypothermia deepens.


Reduced left ventricular diameters at onset of bradycardia during epidural anaesthesia.

Pathophysiologic mechanisms of bradycardia during epidural anaesthesia (L3-L4 with 1% lidocaine, 38 ml) were evaluated by studying changes in selected cardiovascular and hormonal parameters. Six of eight subjects (analgesia to T8-T10) remained circulatory stable with no significant changes in heart rate (HR), mean arterial pressure (MAP) and thoracic impedance (TI). In one of two subjects MAP decreased after 25 min from 85 to 50 mmHg (11.3 to 6.7 kPa), HR from 80 to 45 beats.min-1 while thoracic impedance increased from 25.5 to 26.5 ohm. End-systolic diameter (ESD) and end-diastolic diameter (EDD) of the left ventricle determined with echocardiography were reduced from 3.8 to 3.2 cm (17%) and 5.6 to 5.0 cm (11%), respectively. In the other subject MAP decreased after 25 min from 75 to 50 mmHg (10.0 to 6.7 kPa) and HR from 82 to 60 beats.min-1 while thoracic impedance increased from 28.8 to 29.6 ohm. ESD was reduced from 3.8 to 3.3 cm (13%), and EDD from 5.6 to 5.0 cm (11%). Both subjects recovered after infusion of saline and being placed in the head-down position. There were no consistent changes in plasma catecholamines, whereas pancreatic polypeptide increased from 5 and 3 to 152 and 69 pmol.l-1, vasopressin from 3 and 2 to 152 and 46 pmol.l-1, and aldosterone from 282 and 229 to 383 and 485 pmol.l-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Direct spinal effect of intrathecal acetaminophen on visceral noxious stimulation in rabbits.

The aim of this study was to investigate the effect of intrathecal acetaminophen on visceral and somatic noxious stimulation in the intact, non-anesthetized rabbit. Sixteen rabbits had intrathecal catheters implanted surgically. Visceral noxious stimulation was induced by intestinal distension of the distal colon and somatic stimulation with increasing electrical current through skin electrodes placed in either the cervical or the lumbar area. The effect on visceral noxious stimulation was assessed following intrathecal injection of 0.5, 2.5 and 5 mg of acetaminophen and following 10 and 50 mg acetaminophen intravenously. Naloxone 0.2 mg and yohimbine 0.1 mg were administered intrathecally prior to intrathecal injection of acetaminophen 5 mg. A dose-dependent effect of intrathecal acetaminophen against the visceromotor reflex produced by intestinal distension was shown. No effects on thresholds to lumbar or cervical electrical stimulation or intestinal distension were observed following i.v. administration. Thresholds to noxious electrical stimulation were only significantly elevated at the lumbar level following i.t. injection of 5 mg acetaminophen. Naloxone failed to antagonize the effect of intrathecal acetaminophen, whereas intrathecal yohimbine attenuated the effect of intrathecal acetaminophen in both tests. In conclusion, a spinal, dose-dependent, naloxone-irreversible, and yohimbine-reversible effect of intrathecal acetaminophen on electrical and visceral noxious stimulation was demonstrated.


Cardiac herniation after pneumonectomy.

Cardiac herniation is a rare, lethal complication of intrapericardial pneumonectomy demanding urgent treatment. It usually occurs within the first 24 h postoperatively. We present a patient with late cardiac herniation, 28 h postoperatively, where the electrocardiographic changes were evident several hours before the clinical picture became clear. After the diagnosis was made, the patient was treated surgically and survived.


Alfentanil infusion in total intravenous anaesthesia (TIVA). Population pharmacokinetics fails to predict plasma concentration of alfentanil.

In order to evaluate the ability to predict plasma concentrations from population-corrected pharmacokinetics, a prospective study comprising 14 women admitted for elective hysterectomy was done. Alfentanil was given in combination with propofol in a total intravenous anaesthesia technique. The predicted median alfentanil concentration (289(256-363) ng ml-1) was significantly lower than the measured median plasma concentration of 368(168-666) ng ml-1). In conclusion, population-based pharmacokinetics were found not to be accurate as they underestimate plasma concentrations of alfentanil.


Organic structures of the hypercalcified peritubular matrix in horse dentine.

EDTA-insoluble organic structures of the hypercalcified peritubular matrix (PM) in horse dentine were observed by scanning electron microscopy. The PM was enveloped in double cylindrical structures composed of fibrillar sheaths in the inner and outer peripheries. Between the outer fibrillar sheath and intrinsic fibrils of the intertubular matrix, a calcified cementing membrane existed. Within the PM, warped cone-shaped structures of fibrillar sheaths, overlapping at intervals of 4-6 microns and semiconcentrically surrounding the dentinal tubule, extended from the inner fibrillar towards the outer fibrillar sheath. The cone-shaped fibrillar sheaths following the inner and outer fibrillar sheaths were identified as the incremental lines of the PM. Most of these fibrils may be collagen although it could not be confirmed, whereas non-collagenous organic materials in the lateral branches of the dentinal tubule are radially arranged in the PM. These EDTA-insoluble structures were three-dimensionally illustrated using an image-analysing system.


Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.


Growth of the perichondrium and the chondroepiphysis: experimental approach in the rat proximal tibial epiphysis.

In the present paper we study the participation of the perichondrium in chondroepiphysis development analyzing its in vitro growth pattern without the perichondrium. We also advance the descriptive morphological results. We have observed that the chondroepiphysis without perichondrium grew with an almost normal pattern. Most of the cells that participated in chondroepiphysis growth came from the lateral region of the growth plate.


Fractal dimensions of cranial sutures and waveforms.

Two quite different shapes of cranial sutures ostensibly yield fractal dimensions. The rare, intricate sutures yield the more valid fractal dimensions because self-similar scaling provides a double-log plot of negative slope. These sutures are fractals over a range of several r values. Some of the highly folded, wavy sutures in humans also fill space except at very tiny values of r, but are nonfractal. A great deal depends on whether the dimension D is > 1 and by how much, whether the curve yields a false fractal dimension, whether the curve scales and shows self-similarity, and whether the scaling occurs regularly in the same pattern. We suggest careful attention to the inverse power law equations, which when misused can yield false fractal values. Cranial sutures vary from the simple wavy sutures to the complex folded ones, and, in rare instances, evolve and develop to the self-similar, scaling, elaborate ones called intricate sutures. The main thing is to express the biology precisely, whether waveform regularity or irregularity or scaling elaboration conserving space and the original shape. D values may not in themselves reliably allow such a distinction, by whatever method used.


Human sperm surface mapping with lectins.

Ten fluorescein isothiocyanate (FITC)-linked lectins [Bauhimia purpurea, Concanavalin A, Dolichos biflorus (DBA), Griffonia simplicifolia I, Griffonia simplicifolia II, Maclura pomifera, Arachis hypogea (PNA), Glycine max, Ulex europaeus (UEA) and Triticum vulgaris agglutinin] have been used to study their binding features on the human ejaculate spermatozoa. Qualitative changes in the labeling pattern have been observed in unfixed and acetone-treated spermatozoa. Furthermore, ultrastructural localization of some of the colloidal gold-linked lectins, namely PNA, UEA and DBA, has been attempted to delineate the binding domains of the specific sugars on the sperm surface. It needs to be emphasized that flow-cytometric methods employed in our study, which provide quantitative slant to qualitative data, should be utilized to evaluate the functional status of the spermatozoa.


Clomiphene citrate alters vaginal surface morphology in cycling rats.

Cycling virgin female rats were treated with clomiphene citrate (CC) during dioestrus of the reproductive cycle. Animals were sacrificed 2 days after the initial injection and their vaginal tissue was examined by scanning electron microscopy. Animals treated with one dose of CC had an epithelium consistent with a prolonged dioestrus. Treatment with CC for 2 days induced changes in the epithelium that had no resemblance to any normal hormonally controlled event in the vagina. It was found that CC had effects consistent with progesterone alone as well as effects unique to this superovulatory drug.


Ultrastructure and collagen composition of the myo-fascial junction in rat calf muscles.

The collagen composition and ultrastructural organization of the myo-fascial junction was studied using immunohistochemical techniques and electron microscopy. At the myo-fascial junction, a small amount of type-III collagen was found; however, the major collagen component was the type-I collagen. On the longitudinal sites of muscle cells, there were deep recesses and, within these, finger-like structures containing sarcomeres. In these recesses and in the finger-like structures of the myo-fascial junction, the sarcolemma was thickened (three times) in a similar way to the basal lamina at the myotendinous junction. Thin collagen fibers were closely associated with the thickened sarcolemma of the finger-like structures.


Abdominal part artery of axillary artery: proposed term for the artery supplying the abdominal part of the musculus pectoralis major.

In 1976, the authors reported that the abdominal part artery (Pab) supplying the abdominal part of the pectoralis major muscle usually originates from the axillary artery (Ax). The findings in the present study show that the type of origin of this artery most frequently encountered is type 2-a (44.0%) in which the Pab, as an independent branch (type a), branches out of the second part of the Ax (type 2). The second and third most frequently encountered types are type 2-b (17.0%), where the Pab has a common trunk with the thoracoacromial artery, and type 2-c (10.0%), where it has a common trunk with the lateral thoracic artery. By classification according to the supplying areas, 67% was type I-B, supplying the lower part of the pectoralis minor muscle and the abdominal part of the muscle. In 5%, the branch as type I-A courses down to the sternocostal part. In most cases (types A and B in 91%), this artery originates from the Ax proximal to the ansa mediana of the brachial plexus; however, in 4% providing the superficial brachial artery, the Pab branches out from the superficial brachial artery. Based on those findings, the authors would propose that the artery be named the arteria partis abdominalis or Pab.


In vitro effects of elastase on rabbit craniomandibular joint articular disks.

This in vitro study correlates morphologic and radioimmunoassay (RIA) findings on the effects of elastase on the elastic fibers that are found in the rabbit craniomandibular joint (CMJ) articular disk. Articular disks were removed from rabbit CMJs at necropsy, and cut sagittally into two pieces which were incubated in 0.3 ml of phosphate-buffered saline containing either 0, 12.5, 25 or 50 units of porcine pancreatic elastase for either 1, 3 or 24 h. The quantitative RIA findings correlated well with the qualitative light-microscopic observations in that both methods showed a reduction in the amounts of elastin in the CMJ disks following enzyme treatment. However, the morphologic appearance of most of the elastase-treated disks suggested that the destruction of the elastic fibers was more extensive than was suggested by the results of the RIA which indicated that some elastin remained in the tissues of the disks even when the highest enzyme level and longest incubation period were combined. The results of this study also support the interpretation that the resorcin-fuchsin-stained fibers in the rabbit CMJ disk are elastic fibers.


Biogenesis of the ciliary roots: participation of the dictyosomes of Golgi's complex.

In this study, the hypothesis of a possible biogenesis of the ciliary roots is suggested, after observing the cilia neurons under the electron microscope, which were found as an exception in the periaqueductal nucleus of the mesencephalon in the domestic cat, conserving the potential to differentiate the cilia, basal bodies and ciliary roots. The dictyosomes of Golgi's complex and Golgi's vesicles participated in this biogenesis. Vesicles of approximately 71.6 nm in diameter had become separated from the periphery of the flattened discoid cisterns of the dictyosome and were aligned normally, in tangential contact with each other, forming rows of vesicles or 'ringed chains', whose points of contact formed the beginning of the 'periodic striation' of a thin ciliary root. Later, the lateral walls of the vesicles and the molecules of the intracisternal proteins gave rise to the interperiodic microfilaments, when the carrier proteins were transformed into structural proteins of the ciliary roots. The parallel apposition of several ringed chains or thin ciliary roots, with their rings joined at the same level (or transversal striations), gave rise to thicker striated roots. This hypothesis of an ultrastructural biogenesis of the striated ciliary roots involves the following six stages: stage I = separation of Golgi's vesicles from the periphery of the flattened disk of dictyosomes near the basal body, with a diameter of over 71.6 nm; stage II = reinforcement of the membrane of the vesicles at the two opposite polar ends of its larger diameter; stage III = alignment of vesicles to form ringed chains, due to the tangential contact between their reinforced points; initiation of the 71.6-nm striation period, roots ringed linearly; stage IV = formation of joining microfilaments between periods (69.2 nm) with the lateral walls of the vesicles and the molecules of the proteins in their content; stage V = lengthening of the thin ciliary roots due to the coupling of new Golgi's vesicles at their ends so that their length increases as a result of the addition of terminal vesicles; stage VI = increase in thickness of the thin ciliary roots, due to the parallel apposition of several ringed chains or thin ringed ciliary roots, at the point where their transversal striation points coincide.


Branches of the right pulmonary artery supplying the basal segments of the lung.

The studies were carried out on 100 right lungs taken from dead human bodies of both sexes whose age varied from 16 to 81 years. The pulmonary artery and the bronchus were injected with a 65% solution of duracryl and then digested in sulfuric acid. The specimens obtained were then examined to determine the number and dimensions of the branches of the basal portion of the right pulmonary artery (RPA) penetrating into the basal segments of the right lower pulmonary lobe. Their length was 52 mm at the most, and their diameter 14 mm. Three types of ramification of the basal portion of the RPA were distinguished on the basis of the trunks, segmental and subsegmental branches present. In 72% of the cases the branches penetrating into the basal segments showed a tree-like type, in 2% of the cases a bushy-like type and in 26% of the cases a middle type.


Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.


Fibroblast growth factor: effects on osteogenesis and chondrogenesis in the chick embryo.

In vivo effects of fibroblast growth factor (FGF) on osteogenesis were evaluated in the chick embryo. Autoradiographic studies of 3H-proline labeling over bone matrix indicated that 24 h after treatment on day 11, FGF stimulated osteogenic cell proliferation, while inhibiting the production of bone matrix collagen. However, 4 days after multiple doses of FGF, the large pool of newly formed osteogenic and chondrogenic cells expressed their function with the increased formation of matrix. The data provide in vivo evidence of the effects of exogenous FGF on osteogenesis and also point to a possible role for FGF both in embryonic osteogenesis and in fracture repair.


Quantification and distribution of troponin-T in cultures of chick embryo myocardiocytes.

We analyzed the distribution and labeling patterns of troponin-T, a protein involved in the regulation of striated muscle contraction, in myocardiocytes obtained from chick embryos in Hamburger and Hamilton's stage 25 and 39, and cultured for 8 days. Troponin-T expression was examined with indirect immunofluorescence, densitometry, fluorescence-activated cell sorting, electrophoresis and immunoblotting. The patterns of expression of troponin-T were compared with those of actin to determine possible correlations in different stages of chick embryo development and culture. Our findings show that in both stages of embryonic development, the cellular accumulation of troponin-T changed after 8 days of culture. Our results revealed a quantitative modification with time: after 4 days of culture there was a significant increase in this protein, followed by a slight additional increase after 8 days of culture. Flow cytometry findings confirmed these trends over time, showing a significant increase in positive cells after 4 days, followed by a smaller rise after 8 days of culture. In comparison with actin, this pattern was similar only in cells from Hamburger and Hamilton's stage 25 embryos.


Changes in glycosaminoglycan binding to collagen during desmoid mineralization as revealed by different electron-microscopic staining techniques.

Mineralization at collagen fibrils is regulated by glycosaminoglycans (GAG). Alterations in proteoglycan composition during mineralization as well as inhibition of mineralization by GAGs are well documented. Collagen-GAG interactions during desmoid osteogenesis in fetal rat calvariae were investigated ultrastructurally by means of different fixation techniques. Mineralization was restricted to the collagen of the osteoid at the ectocranial side. Beyond the osteoid, one layer containing degenerated cells was found, followed by sheets of healthy osteoblasts with nonmineralized collagen fibrils. These fibrils were ordered in bundles, but were irregularly arranged in the mineralized osteoid. After fixation in glutaraldehyde-ruthenium red (GA-RR), small RR-positive granules were periodically attached to the fibrils of the nonmineralized collagen. These granules were absent at collagen in the mineralized osteoid. Periodically bound granules (periodicity of 62 nm) could clearly be demonstrated along collagen fibrils by pretreatment with the positively charged protamine sulfate and subsequent fixation in GA-RR in the nonmineralized collagen. In the mineralized osteoid, however, these granules were present, but periodic binding was missing. Heparin pretreatment followed by fixation in GA-RR revealed periodically bound fine strands between collagen fibrils running parallel in the nonmineralized collagen; these threads were absent in the mineralizing osteoid. Restriction of mineralization to osteoid at the mineralization border may be reflected by the observed changes in GAG binding to collagen fibrils within the osteoid of developing fetal calvariae in contrast to binding to collagen in nonmineralized areas.


Quantification of thymic innervation in juvenile and aged rats.

Thymic innervation has been quantified using silver impregnation and light microscopy on 5 juvenile and 5 aged rats. There is an increase in innervation density of the thymus with age, exceeding the reduction of thymic volume due to involution. The density of innervation correlates inversely with thymic volume. From previous studies, we conclude that thymic innervation is confined to the extraparenchymal compartment. Ultrastructural morphometry shows this compartment to maintain its volume during involution of the remaining thymic tissue. Related only to the extraparenchymal compartment, the innervation increases by a factor two with age.


Immunohistochemical and ultrastructural examination of histiocytosis X cells in pulmonary eosinophilic granuloma.

We describe histological, immunohistochemical, and ultrastructural findings in pulmonary eosinophilic granuloma (PEG) from three patients. The specimens were taken by open-lung biopsy. The lesions of the lung were composed of histiocytic cells, macrophages and eosinophils. The histiocytic cells reacted positively with anti-S-100 protein antibody. The histiocytic cells had various types of Birbeck granules in the cytoplasm. The histiocytic cells were histiocytosis X (HX) cells considered to be derived from Langerhans cells. Sporadic mitosis of HX cells was observed. Some HX cells had migrated from the lesions into the alveolar spaces through the alveolar cell layers. In the lesions of PEG, HX cells have self-reproduction and migration capability.


Spontaneous rupture of aortic arch through an atheromatous plaque resulting in pseudoaneurysm.

An autopsy case of spontaneous rupture of aortic arch through an atheromatous plaque which resulted in pseudoaneurysm is reported. The aorta was involved by severe atherosclerosis with scattered calcifications over the entire surface, and there was a fusiform aneurysm protruding from a transmural tear in the aortic arch. Histopathologically, no evidence of dissection or inflammatory change was noted in the aorta, and no histologic component of the aortic media was observed in the aneurysmal wall. However, degenerative change of cystic and laminar medial necrosis were recognized near the ruptured site. Such localized degenerative change was considered to be caused by circulatory disturbance which originated from disruption of the vasa vasorum due to aortic rupture.


Extremely well differentiated papillary adenocarcinoma of the lung with prominent cilia formation.

We describe a 54-year-old woman with primary pulmonary adenocarcinoma showing a characteristic papillary architecture and prominent cilia formation. Immunohistochemically, the tumor cells were positive for carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and Leu-M1, and negative for lactoferrin and surfactant apoprotein. An ultrastructural study also indicated differentiation toward bronchial surface epithelial cells. To our knowledge, this type of neoplasm has not been reported as peripheral-type adenocarcinoma of the lung.


True pulmonary carcinosarcoma (squamous cell carcinoma and chondrosarcoma). A case report.

True pulmonary carcinosarcoma (squamous cell carcinoma and chondrosarcoma) originating in the right lower lobe in a 62-year-old Japanese male is reported. The tumor, measuring 5.5 x 3.5 x 3.5 cm, was markedly necrotic and its apex protruded into the bronchial lumen. Light microscopy showed that the tumor was composed of squamous cell carcinoma with sarcomatous spindle or polygonal cell proliferation and true chondrosarcoma. Immunohistochemically, the cytoplasm of numerous cells of the squamous cell carcinoma component was stained with anti-cytokeratin (PKK 1) and the cytoplasmic membrane with anti-epithelial membrane antigen (EMA). Although sarcomatous regions were stained with anti-vimentin (vimentin) and no tumor cells were reactive for EMA, a few tumor cells were positive for PKK 1. The cytoplasm of numerous chondrosarcoma cells was positively stained for vimentin and S-100 protein. Based on these findings, we concluded that the present tumor was a true carcinosarcoma composed of squamous cell carcinoma with sarcomatous regions and true chondrosarcoma.


A case of primary sarcoma of the pulmonary artery.

A 39-year-old male was admitted complaining of nonproductive cough and dyspnea on exertion. Death occurred eight months after onset of the symptoms. Autopsy examination showed that the pulmonary trunk and left main pulmonary artery were markedly dilated and completely occluded by a tumor. The tumor had infiltrated into the left upper lobe and mediastinal lymph nodes, and metastatic nodules were found in both lungs and in the left adrenal gland. Small foci of infarction were noted in the lower lobes of both lungs. The tumor cells were of two types; pleomorphic spindle cells and bizarre multinucleated giant cells. Immunohistochemically, they were positive for vimentin, myosin, and lysozyme, but negative for desmin and muscle-specific actin. The cytoplasm of the tumor cells was showed by electron microscopy to contain microfilaments, dense bodies, and pinocytotic vesicles. We diagnosed this case as undifferentiated sarcoma of the pulmonary artery. Approximately 100 cases of pulmonary artery sarcoma have been reported. Histopathologically, almost all of the reported cases showed both spindle cells and pleomorphic giant cells, indicating a biologically anaplastic neoplasm.


A case report of inflammatory pseudosarcoma of the urinary bladder.

A case of inflammatory pseudosarcoma of the urinary bladder in a 35-year-old Japanese male is presented. This benign lesion can easily be mistaken for spindle cell sarcoma since it consists of rhabdomyoblast-like elongated strap cells showing infiltrative growth, and whether it is benign or malignant is difficult to determine by microscopic examination. In this case, spindle cell proliferation extended among bundles of the superficial muscle layer. However, no abnormal mitoses, severe nuclear atypia or cellular pleomorphism could be seen, thus indicating inflammatory pseudosarcoma. Although the lesion was not completely resected, no recurrent disease has been clinically observed for two years following transurethral resection. Urologists and surgical pathologists must be able to detect this lesion in order to avoid unnecessary surgical procedures.


Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases.

We measured type I procollagen carboxyl-terminal propeptide (PICP) by a commercial radioimmunoassay and amino-terminal propeptide (PINP) by an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory in serum from 75 normal women, 10 growing girls, 84 normal men, and 197 patients with various metabolic bone diseases. The molar concentrations of serum PINP were 100-fold higher than those of PICP, suggesting differences in the metabolism of PICP and PINP. In normal women, serum PICP values correlated positively with age and serum PINP values correlated negatively with age (r = 0.28 and -0.32, respectively; P = 0.02). In normal men, however, PICP correlated negatively with age (r = -0.32, P = 0.003) whereas PINP did not change. As assessed by Z scores (SD from age- and sex-specific predicted normal mean), changes in serum PICP and PINP values were concordant in hypoparathyroidism (mean Z scores for PICP and PINP, -0.63 and -1.48, respectively) and Cushing's syndrome (0.50 and 0.40) but were discordant in acromegaly (0.78 and -0.68), hyperthyroidism (1.33 and -0.66), untreated postmenopausal osteoporosis (-0.11 and 0.40), fluoride-treated postmenopausal osteoporosis (-0.61 and 1.08), Paget's disease (4.05 and -0.20), and chronic renal failure (1.45 and -0.50). With either assay, deviations from normal were less pronounced than the deviations of concurrently measured serum osteocalcin and bone alkaline phosphatase values. The deviations in these latter two values agreed better with those of PICP than with those of PINP, except in untreated or fluoride-treated osteoporotic patients.(ABSTRACT TRUNCATED AT 250 WORDS)


A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linked N-telopeptides in urine.

Peptides of low molecular weight that contain pyridinoline cross-links were isolated from adolescent human urine. A fraction was selected that was enriched in the N-telopeptide-to-helix intermolecular cross-linking domain of bone type I collagen. Mouse monoclonal antibodies were generated against these urinary peptides conjugated to a carrier protein as immunogen. A high-affinity antibody was identified that specifically bound to the trivalent peptides derived from the N-telopeptide-to-helix pyridinoline cross-linking site in type I collagen of human bone. This was confirmed by the direct isolation from human bone collagen of similar fragments recognized selectively by the antibody. A sensitive inhibition ELISA was established on microtiter plates that could quantify the bone-derived peptides in human urine. The assay, which can be run directly on untreated urine, was thoroughly tested against samples from normal subjects and from patients with metabolic bone disease. For example, strong correlations with urinary hydroxyproline and total pyridinoline cross-links were found in patients with Paget's disease of bone. The method shows considerable promise as a rapid and specific index of human bone resorption rates, with greatly improved specificity and convenience over total pyridinoline analysis. Potential applications include the study of normal metabolism, the diagnosis and monitoring of bone disease, and evaluating the effectiveness of antiresorption therapies.


Vertebral deformity in men.

Vertebral fracture is the most prevalent manifestation of osteoporosis in women, but there is very little information concerning vertebral fracture in men. These studies begin to determine the prevalence, radiographic character, and relationship to bone mineral density of vertebral deformity in men. A group of 144 white men aged 34-94 years (83% between 50 and 80 years) were studied. Thoracic and lumbar spine radiographs were obtained using standardized techniques, and morphometric measures of vertebrae (T6-L5) were obtained using a computerized digitization pad. Vertebral deformities (wedge, midbody, and crush) were identified using several criteria. In addition, a skeletal radiologist independently identified vertebral deformities, as well as vertebrae affected by epiphysitis (Scheuermann's disease), using classic radiographic criteria. Bone mineral density was measured at lumbar spine and proximal femoral sites using dual-photon absorptiometry. The prevalence of vertebral deformity was related to the criteria used for their identification. Utilizing vertebral-specific criteria (anterior/posterior or midbody/posterior vertebral height more than 3 SD below vertebral specific mean), 10% of subjects had vertebral deformity. Wedge deformity occurred primarily in thoracic vertebrae and were more common than midbody deformity, which occurred more commonly in lumbar vertebrae. Crush deformities were not observed. Evidence of vertebral epiphysitis was present in 9% of subjects but was not responsible for vertebral deformity sufficient to be falsely identified using the more than -3 SD criterion. Bone mineral density in subjects with vertebral deformity was clearly reduced at both vertebral (p = 0.003) and proximal femoral (p = 0.002) measurements sites. The number of vertebral deformities was negatively correlated with vertebral bone mineral density.(ABSTRACT TRUNCATED AT 250 WORDS)


Do estrogens improve bone mineral density in osteoporotic women over age 65?

A retrospective analysis of our experience with estrogen and fluoride treatment in 91 patients with postmenopausal osteopenia followed for 6-47 months has been performed. Treatment included calcium (1000 mg/day) and either conjugated estrogens (0.625 mg/day) or sodium fluoride (50 mg/day), or both. All patients had at least two serial dual-photon spinal bone mineral density measurements performed 6 months or more apart. Estrogen treatment was associated with increased bone mineral density (5.3%/year), as was fluoride alone (7.5%/year). Estrogen and fluoride together were additive (9.6%/year). In women over age 65 the estrogen effect was just as great (6.9%/year) as in younger women. Estrogen benefit occurred predominantly in the first 18 months of treatment (7.0%/year), after which time changes in bone mineral density were similar to those in untreated controls, who showed stable bone mineral density. We conclude that aggressive estrogen and fluoride treatment tailored to the severity of the individual's postmenopausal osteopenia results in short-term improvement in spinal bone mineral density. These data further support that elderly women respond to estrogen replacement therapy with absolute and relative increments in bone density similar to those in younger women.


Bone mineral density, muscle strength, and recreational exercise in men.

Muscle strength has been shown to predict bone mineral density (BMD) in women. We examined this relationship in 50 healthy men who ranged in age from 28 to 51 years (average 38.3 years). BMD of the lumbar spine, proximal femur, whole body, and tibia were measured by dual-energy x-ray absorptiometry (Hologic QDR 1000W). Dynamic strength using one repetition maximum was assessed for the biceps, quadriceps, and back extensors and for the hip abductors, adductors, and flexors. Isometric grip strength was measured by dynamometry. Daily walking mileage was assessed by 9 week stepmeter records and kinematic analysis of video filming. Subjects were designated as exercisers and nonexercisers. Exercisers participated in recreational exercise at least two times each week. The results demonstrated that BMD at all sites correlated with back and biceps strength (p < 0.01 to p = 0.0001). Body weight correlated with tibia and whole-body BMD (p < 0.001); age negatively correlated with Ward's triangle BMD (p < 0.01). In stepwise multiple regressions, back strength was the only independent predictor of spine and femoral neck density (R2 = 0.27). Further, back strength was the most robust predictor of BMD at the trochanter, Ward's triangle, whole body, and tibia, although biceps strength, age, body weight, and leg strength contributed significantly to BMD at these skeletal sites, accounting for 35-52% of the variance in BMD. Exercisers and nonexercisers were similar for walking (3.97 versus 3.94 miles/day), age (37.8 versus 38.5) years, and weight (80.0 versus 77.7 kg). However, BMD and muscle strength were significantly greater in exercises than in nonexercisers.(ABSTRACT TRUNCATED AT 250 WORDS)


Diurnal variation in serum markers of type I collagen synthesis and degradation in healthy premenopausal women.

There are several indications that the functions of human osteoblasts and osteoclasts have circadian rhythms with peak activities occurring at night. It is not known, however, whether the principal function of these cells, namely synthesis and degradation of the organic matrix of bone, of which about 90% is type I collagen, also has a circadian rhythm. This was therefore investigated for both the formation of type I collagen and the degradation of type I collagen in bone using two newly developed serum markers: the serum concentration of the carboxy-terminal propeptide of type I procollagen (PICP) as a marker of formation and the serum concentration of the carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) as a marker of degradation. PICP and ICTP were measured by RIA in samples taken every 3 h over a 24 h period in 12 healthy premenopausal women (age 32 +/- 5 years, mean +/- SD). Both PICP (p = 0.003) and ICTP (p = 0.00003) showed a significant circadian rhythm, with about 20% higher values at night than in the afternoon. We conclude that serum markers of both the formation of new type I collagen and the degradation of old type I collagen in bone exhibit a clear circadian rhythm, with increased activity of both osteoblasts and osteoclasts at night. The etiology of this circadian rhythm is still unknown.


Regulation of cytoplasmic calcium concentration in tetracycline-treated osteoclasts.

The ability of low-dose tetracyclines to inhibit collagenase activity and inactivate osteoclasts suggests that these compounds have great potential as a prophylaxis for metabolic bone disease. However, the cellular mechanism by which tetracyclines interact with skeletal tissue is not yet clear. To better understand the effects of tetracyclines on bone metabolism, we examined their effect on osteoclast activity in vitro. Because tetracyclines can enter the cell and bind calcium and have been reported to directly interact with osteoclasts, we postulated that exposure to either of two tetracyclines, minocycline or doxycycline, would alter cytosolic Ca2+ regulation in rat osteoclasts. [Ca2+]i was measured in single rat osteoclasts utilizing fura-2. Addition of extracellular Ca2+ (5 mM CaCl2), a potent osteoclast inhibitor, increased [Ca2+]i in all osteoclasts, but 10(-6) M salmon calcitonin (sCT) did so only in a subpopulation of osteoclasts. Neither minocycline nor doxycycline (10 micrograms/ml) altered steady-state osteoclast [Ca2+]i. Further, neither minocycline nor doxycycline pretreatment affected the sCT-mediated increases in [Ca2+]i. However, tetracycline pretreatment significantly decreased the cytosolic Ca2+ response to extracellular CaCl2. Our results strongly suggest that tetracyclines have a specific effect on extracellular Ca(2+)-stimulated cytosolic Ca2+ mobilization in osteoclasts, which is not solely dependent on their ability to buffer Ca2+. Furthermore, these results point to the potential use of tetracyclines as probes to study cytosolic Ca2+ regulation. However, that tetracyclines attenuate a signal response associated with decreased osteoclastic resorption suggests that the reported antiresorptive attributes of tetracyclines must be achieved independently of an effect on osteoclastic cytosolic Ca2+.


Interrelationship between parathyroid hormone and insulin: effects on DNA synthesis in UMR-106-01 cells.

UMR-106-01 osteoblast-like cells respond to high concentrations of parathyroid hormone (PTH) in vitro by decreasing thymidine incorporation, a marker of DNA synthesis and cell proliferation. This response is different from in vivo conditions, such as primary and secondary hyperparathyroidism, in which high PTH levels are associated with an increased number of osteoblasts. When the response of UMR-106-01 cells to PTH is evaluated in vitro, however, these cells are exposed to only a single hormone. The present study was designed to evaluate the combined effects of two hormones, PTH and insulin, on the DNA synthesis of UMR-106-01 cells. PTH is known to decrease and insulin to increase thymidine incorporation by UMR-106-01 cells. To examine the interaction of these hormones, acute studies, defined as a 24 h exposure to hormone, and chronic studies, defined as a 7 day exposure to hormone, were performed. Both acute and chronic exposure to 10(-9) M PTH decreased thymidine incorporation by UMR-106-01 cells, with suppression ranging from 27 to 81% (P < 0.05). Both acute and chronic exposure to 10(-8) M insulin (INS) increased thymidine incorporation by UMR-106-01 cells; this ranged from 26 to 58% (P < 0.05). However, chronic exposure to 10(-9) M PTH followed by an acute exposure to 10(-8) M INS resulted in a 710% increase in thymidine incorporation (P < 0.01). Reversing the sequence by chronically exposing UMR-106-01 cells to 10(-8) M INS followed by acute exposure to 10(-9) M PTH resulted in a 53% decrease in thymidine incorporation (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo assessment of forearm bone mass and ulnar bending stiffness in healthy men.

The cross-sectional bending stiffness EI of the ulna was measured in vivo by mechanical resistance tissue analysis (MRTA) in 90 men aged 19-89 years. MRTA measures the impedance response of low-frequency vibrations to determine EI, which is a reflection of elastic modulus E and moment of inertia I for the whole ulna. EI was compared to conventional estimates of bone mineral content (BMC), bone width (BW), and BMC/BW, which were all measured by single-photon absorptiometry. Results obtained from the nondominant ulna indicate that BW increases (r = 0.27, p = 0.01) and ulnar BMC/BW decreases (r = -0.31, p < or = 0.005) with age. Neither BMC nor EI declined with age. The single best predictor of EI was BW (r2 = 0.47, p = 0.0001), and further small but significant contributions were made by BMC (r2 = 0.53, p = 0.0001) and grip strength (r2 = 0.55, p = 0.0001). These results suggest that the resistance of older men to forearm fracture is related to age-associated changes in the moment of inertia achieved by redistributing bone mineral farther from the bending axis. We conclude that the in vivo assessment of bone geometry offers important insights to the comprehensive evaluation of bone strength.


Kallikrein-like activity in nonpregnant and pregnant rat uterus, fetal membranes, placenta and amniotic fluid.

SBTI-resistant kininogenase activity was found in nonpregnant and pregnant rat uterus, placenta, amniotic fluid and fetal membranes. After trypsin treatment the kininogenase activity increased 2-3 fold. Total kininogenase (active plus inactive) was completely blocked by kallikrein antibodies. The physiological role of these kallikrein-like enzymes is unknown. It is speculated that these enzymes play a local role, perhaps in the processing of polypeptide hormones of through the release of kinins in the regulation of uterine blood flow.


Dynamics of kallikrein activity in different biological fluids of pregnant animals.

Kallikrein activity was measured in urine, saliva and blood plasma samples from pregnant cows by using the chromogenic substrates S-2266 and S-2302 (Kabi, Sweden). It was found that the kallikrein activity in these biological fluids showed a trend to an increase with a peak prior to parturition. At the same time the plasma prekallikrein levels decreased during pregnancy reaching the lowest values at the end of the gestation period.


Bovine pancreatic kallikrein: purification and radioimmunoassay.

Kallikrein was isolated from bovine pancreatic tissue by a sequential procedure of anion-exchange, hydrophobic interaction and gel filtration chromatographies. A monospecific polyclonal antiserum to the pure enzyme was raised in rabbits and was used to set up a specific radioimmunoassay for bovine tissue kallikrein.


Purification and characterization of new arginine esteropeptidase from the soluble fraction of human submaxillary glands.

An arginine esteropeptidase was completely purified from the soluble fraction of human submaxillary glands. The molecular weight was calculated to be 12,000, having 2 species of subunits. The study of the effect of inhibitors confirmed the enzyme's serine protease-like characteristics. The best ester and amide substrates were Tos-Arg-OMe and D-Ile-Pro-Arg-pNA, respectively.


Molecular diversity of tissue kallikrein in human saliva.

The present study was conducted to explore the extent of heterogeneity of tissue kallikrein in saliva using immunological analysis and to demonstrate that such heterogeneity resulted from secretory phenomena and not degradation secondary to secretion. Human mixed saliva was collected by paraffin-stimulation and centrifuged at 10,000 rpm for 10 minutes. Special collectors were used to obtain parotid and submandibular/sublingual saliva using citric acid stimulation. Western blot analysis of human mixed saliva demonstrated major immunoreactive species with molecular masses of < 20 KD, 45 KD, 60 KD, 90 KD and > 200 KD. The polyclonal antibody used for these blotting studies was monofunctional with respect to reaction with purified salivary tissue kallikrein. Only the < 29 KD and 45 KD species were active using an enzyme overlay technique. While a similar distribution of molecular weight material was observed in both parotid and submandibular saliva, the amount of immunoreactive material was markedly less in parotid secretion. In addition, the < 20 KD material was essentially absent in parotid saliva. Treatment of the saliva with thermolysin eliminated the immunoreactive band at 60 KD while treatment with various glycosidases also eliminated some heterogeneity. These results demonstrate considerable variation in tissue kallikrein expression in salivary gland secretions.


Detection and separation of some arginine amidases including tissue kallikrein from human seminal plasma.

A plasminogen/plasmin like substance (AHSAA-1), with affinity to lysine column was separated from DEAE-cellulose adsorbed human seminal plasma. Two forms of acidic arginine amidase with different affinities to LBTI (AHSAA-2) and aprotinin columns (AHSAA-3) were separated from the DEAE-cellulose adsorbed preparation and AHSAA-3 was identified as tissue kallikrein. Two basic arginine amidase preparations having affinity to LBTI (BHSAA-1) and aprotinin column were also separated from the CM-cellulose adsorbed human seminal plasma. Three basic arginine amidases with different molecular mass (BHSAA-2 to 4) were separated by Cellulofine GCL-2000 gel filtration from aprotinin adsorbed material and some of their properties were examined.


Immunoassays for the determination of human tissue kallikrein (TK) in different body fluids based on monoclonal antibodies.

A monoclonal antibody produced against human tissue kallikrein was used to develop solid phase immunoassays for the determination of total immunoreactive tissue kallikrein, of the complex of tissue kallikrein with alpha 1-proteinase inhibitor, and of enzymatically active tissue kallikrein. The assays permit the specific determination of various forms of tissue kallikrein in body fluids and should be very useful in studies on the biological function of tissue kallikrein-kinin systems.


Biological assay for tissue kallikrein: comparison with the synthetic substrate S2266.

A highly sensitive biological assay for tissue kallikrein is described, using human kininogen as substrate; and quantitation, by radioimmunoassay, of generated kinins. Using purified human urinary kallikrein as a reference standard we have correlated the kininogenase activity of kallikrein with amidase activity as measured by cleavage of the synthetic substrate S2266.


Expression of kallikrein-binding protein and alpha 1-antitrypsin genes in response to sex hormones, growth, inflammation and hypertension.

We have recently purified rat kallikrein-binding protein (RKBP) and alpha 1-antitrypsin (alpha 1-AT) to homogeneity and isolated, sequenced cDNAs encoding these potential regulators of tissue kallikreins. Characterization of the cDNA and the gene has established the identity of the kallikrein-binding protein as a new member of the serpin (serine proteinase inhibitor) superfamily. Using the cDNA probes in Northern blot hybridization, we found a differential regulation of RKBP and alpha 1-AT gene expression in the liver. Ovariectomy results in a 67% reduction of RKBP mRNA levels but a 30% increase of alpha 1-AT mRNA levels. Estradiol or progesterone treatment of the ovariectomized rats increases RKBP transcripts by 2.5- and 6.5-fold, respectively, but reduces alpha 1-AT mRNA level by 30% and 45%, respectively. In contrast to kininogen expression, both RKBP and alpha 1-AT mRNA levels in the liver are at the lowest at birth and rapidly increase during growth and development. Rats injected with endotoxin from 4 to 24 h show a time-dependent decrease of RKBP mRNA levels while the same treatment induces alpha 1-AT gene expression. RKBP mRNA levels in the normotensive Wistar Kyoto (WKY) rats are higher than those in the spontaneously hypertensive rats (SHR) while there are no differences of alpha 1-AT mRNA levels between SHR and WKY.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of glandular and plasma kallikrein by benzamidine derivatives.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with other cationic groups are inhibitors of trypsin-like serine proteinases. Particularly aromatic ring structures with an amidino moiety have high affinity for these enzymes. In most cases ordinary benzamidine derivatives are no selective inhibitors, however, among derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-amino-alkylcarboxylic acids selective competitive inhibitors of several enzymes were found. Amides of phenyl-alpha-aminobutyric acid containing an amidino moiety are inhibitors of plasma kallikrein. The p-amidinoanilide of 2-tosylamino-4-phenylbutyric acid inhibits selectively plasma kallikrein with a Ki of 0.70 mumol/l. In contrast, potent and selective inhibitors of glandular kallikrein were hardly found among benzamidines.


A finding of highly selective synthetic inhibitor of plasma kallikrein; its action to bradykinin generation, intrinsic coagulation and experimental DIC.

We found a novel and highly selective synthetic inhibitor of plasma kallikrein (PK), called PKSI-527; the Ki value was 0.81 microM. PKSI-527 inhibited the bradykinin (BK) generation induced by kaolin and prolonged partial thromboplastin time (PTT). PKSI-527 prevented the decrease of fibrinogen (Fg) levels due to i.v. injection of ellagic acid in mice and ameliorated the endotoxin (ET)-induced DIC in rats.


Studies on the design of orally active peptide analogues.

Converting peptide inhibitors of tissue kallikrein into drugs requires, among other things, that the compounds be made orally active. Measurement of the rate at which peptides diffuse across model epithelial membranes that mimic the human gut has allowed a quantitative relationship relating lipophilicity and % absorption to be developed. The absorption of simple peptides from the human gut may be predicted with this equation.


A systematic approach for determining minimum inhibitory sequence and contribution of individual residues in binding of kininogen fragments to tissue kallikrein.

A systematic approach to evaluate the contribution of individual residues occurring within the sequence Ser386-Pro-Phe-Arg-Ser-Val-Gln392 from bovine kininogen towards binding to tissue kallikrein is developed. Of the 21 sequences which can be formed, no dipeptide and only one tripeptide measurably inhibits the enzyme. Almost 80% of the binding energy of the substrate analogue inhibitors comes from the core sequence Phe-Arg-Ser which occurs between P2 and P1'. Molecular models developed from the Chen-Bode coordinates of the aprotinin--beta-PPK complex have been used to interpret the results of these studies.


Characterization of cell binding and thrombin inhibitory regions on kininogens' heavy chain.

Investigations have been performed to determine the domain(s) on kininogens' heavy chain that binds to platelets and contains thrombin inhibitory region Domain 3, but not domains 1 or 2, completely inhibited 125I-HK binding to platelets (Ki = 24 nM +/- 7, n = 4). D3 binding to platelets was saturable with an apparent Kd of 39 nM +/- 8 (n = 4) and 1227 +/- 404 binding sites/platelet. D3 inhibited 125I-thrombin binding to platelets which prevented thrombin-induced platelet aggregation and secretion. These studies indicate that D3 on kininogens' heavy chain contains a cell binding region and another region which inhibits thrombin binding and activation of platelets.


Parallel procoagulant and anticoagulant pathways for high molecular weight kininogen coagulant function.

High molecular weight kininogen (HK) or its procoagulant light-chain but not the heavy chain potentiated the heparin enhancement of antithrombin III inactivation of plasma kallikrein and factor XIa from 10-50-fold to approximately 1000-fold at I 0.15, pH 7.4, 25 degrees C. This potentiation resulted in antithrombin becoming a predominant inhibitor of kallikrein and factor XIa in heparinized normal but not HK-deficient plasmas. The heparin chain-length and salt dependence of this potentiation suggested an anticoagulant action of HK analogous to its procoagulant action.


Purification and some properties of kininogens in canine plasma.

High molecular weight (HMW) and low molecular weight (LMW) kininogens were present in canine plasma, and its content ratio was 1:3. Both kininogens were purified by ion-exchange and affinity chromatographies. Purified HMW kininogen might be heterogeneous molecule. And two LMW kininogens were purified and properties of both LMW kininogens were almost the same.


Evolution of the kallikrein gene family.

All kallikrein-like genes that have been studied to date are composed of 5 exons and the tertiary structures of the encoded enzymes are remarkably similar. In the mouse and rat, these genes are highly conserved, tightly linked and tandemly arranged. In other species, such as the human, the family is less well defined and seems to be much smaller than that of the mouse and rat. Although extensively studied, the exact physiologic significance is not known for many kallikrein gene family members, however, they are thought to play important roles in processing biologically important peptide precursors. Given the potential importance of these mammalian enzymes as a group of highly selective peptide processing enzymes, it would be helpful to know more about the ways in which this family varies from species to species, especially with respect to the size of the family in each species. The evolutionary mechanisms which have shaped this family of genes are largely unknown, however, enough data has been generated to begin understanding the pathway by which this gene family has evolved.


The structure and expression of the genes for T-kininogen in the rat.

T-kininogen plays an important role in the acute phase response to trauma in the rat as a possible source of kinins and as a cysteine proteinase inhibitor. Two T-kininogens are expressed by rat liver from two separate genes. T-kininogen expression in liver during the acute phase response is regulated at the level of transcription. The similarity in T- and K-kininogen gene structure suggests divergent evolution from a common gene ancestor.


Increased uptake of T-kininogen by the liver in inflammatory conditions.

The distribution of [125I]T-kininogen in the liver of rats was found to be increased by laparotomy-, turpentine- or lipopolysaccharide-induced inflammation, whereas no such increase was observed in other organs or when 125I-labeled carboxymethylated T-kininogen, which does not inhibit cysteine proteinase, was used. These results suggest that the liver plays an important role in clearing T-kininogen from the circulation during inflammation.


The role of the kininogens as cysteine proteinase inhibitors in local and systemic inflammation.

The contribution of the kininogens and cystatin C to the functional inhibitory capacity for cysteine proteinases of blood plasma and inflammatory secretions was estimated from ex vivo experiments. 98.5% of the inhibitory capacity of blood plasma for cathepsin L (4-5 microM) is provided by the kininogens ensuring a complete control of this enzyme even at a lowered kininogen concentration. Control of cathepsin B activity by the kininogens is incomplete and depends critically on the active concentration of cystatin C (70 nM in normal plasma), which is reduced in blood plasma of polytraumatized and septic patients and very low in epithelial lining fluid of the shock lung.


Caiman kininogen-like cysteine proteinase inhibitor.

Kininogens are the major mammalian plasma cysteine proteinase inhibitors; a kininogen-like protein was also found in the snake Bothrops jararaca plasma. This communication describes a kininogen-like protein in plasma of Caiman crocodilus vacare. Caiman crude plasma, unlike snake plasma, contains a detectable cysteine proteinase inhibitor. The inhibitor was purified by DEAE-Sephadex ion-exchange chromatography and chromatography on carboxy-methylated-papain-Sepharose. The estimated molecular weight of Caiman cysteine proteinase inhibitor is 70,000. Caiman plasma also hydrolyzes plasma kallikrein synthetic substrates and inhibits trypsin. Reptilian kininogen may lack the site for interaction with plasma prokallikrein, and the sequence of the released kinin may be distinct from bradykinin. The poor effectiveness of bradykinin on reptile smooth muscle shows that the reptile kinin receptors may be adapted to a specific kinin.


Isolation and characterisation of chicken L- and H-kininogens and their interaction with chicken cysteine proteinases and papain.

We have isolated L- and H-kininogens from chicken egg white and plasma using affinity chromatography on CM-papain Sepharose, Con-A Sepharose, gel filtration and ion exchange chromatography, in pure form. In chicken plasma mostly HK was present, whereas in chicken egg white both LK and HK were identified. The determined Mr of LK and HK were 69000 and about 96000, respectively. They occurred in multiple forms with pI 4.3-5.2. The inhibitory activity of chicken HK was tested against cysteine proteinases cathepsins B and L, isolated from chicken liver, and papain. The obtained Ki values demonstrated that chicken H-kininogen is a strong inhibitor of chicken cathepsin L and papain, but much weaker inhibitor of chicken cathepsin B.


A re-evaluation of the tissue-specific pattern of expression of the rat kallikrein gene family.

We have used reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and insitu hybridization to further define the tissue and cell-specific pattern of expression of the rat kallikrein gene family. rKlk1 gene expression has been unequivocally demonstrated in the rat heart, ovary and testis. Similarly, rKlk3, 4 and 9 gene expression has been demonstrated in the testis/ovary, heart and testis respectively. Insitu hybridization has identified the expression of kallikrein gene family members to specific cell-types in the rat ovary (granulosa cell) and testis (germ cell).


Structural features of two kininase I-type enzymes revealed by molecular cloning.

Kininase I-type carboxypeptidases remove a single C-terminal Arg residue from kinins. The circulating kininase I (carboxypeptidase N) contains two types of subunits: a 50 kDa catalytic subunit and an 83 kDa carrier subunit which protects the active subunit in blood. The 83 kDa subunit contains 12 leucine-rich tandem repeats, similar in sequence to other proteins with binding functions. Human carboxypeptidase M is a widely distributed "tissue kininase I" bound to plasma membranes. It has 41% sequence identity with the 50 kDa subunit of carboxypeptidase N and may regulate the activity of kinins and other peptides at the cell surface.


Comparative molecular modeling of the active subunit of human kininase I.

The structure of the enzymatically active subunit of human plasma carboxypeptidase N was determined by computer aided model building by homology using the structural coordinates from carboxypeptidase A. The active site of carboxypeptidase N has been well conserved in comparison with carboxypeptidase A. Differences in substrate specificity can be explained by the comparison of energetically favorable binding sites for different atomic probe groups.


Localization and characterization of human salivary kininases.

The human saliva of normal subjects containing large amounts of basic carboxypeptidase produces decarboxylated non inflammatory peptides, for instance, kinins and anaphilotoxins C3a, C4a and C5a. A reduction of epithelial cell-bonded enzyme (carboxypeptidase M-type or kininase I), produces inflammations by the active intact kinins and the initiation of the alternative activating pathway of complement by active anaphilotoxins, which generate complement cleavage products, containing potential destructive mechanism.


Human serum carboxypeptidase U: a new kininase?

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its instability at room temperature and at 37 degrees C, we named it "unstable carboxypeptidase" (carboxypeptidase U, CPU). The enzymatically active subunit of carboxypeptidase U was purified and exhibits a single band at 53 kDa on SDS-PAGE.


Functional diversity of proteinases encoded by genes of the rat tissue kallikrein family.

A group of proteinases closely related to tissue kallikrein was purified from the rat submandibular gland. Physicochemical characterization of these proteinases, including amino terminal sequencing, allowed correlation with the genes of the rat kallikrein family. In spite of their similar structure, these proteinases have different substrate specificities and different susceptibilities to inhibitors which suggest that they do not share the same biological function. Kallikrein-like proteinases also have restricted specificities that are probably related to their extended substrate binding site. This makes them good candidates for processing inactive protein or peptide precursors into biologically active peptides. A general approach to identifying the putative biological substrates of individual proteinases based on analysis of the specific cleavage of synthetic and natural peptide substrates by kallikrein-related proteinases is described.


A useful and sensitive method for demonstration of the involvement of either kallikrein-kinin system in pathological states.

In rat carrageenin-induced pleurisy, bradykinin (BK) was hardly able to be detected (< 160 pg per rat) in the exudates. In contrast, des-Phe8-Arg9-BK (des-8,9-BK) level, determined by an enzyme immunoassay (EIA) newly developed, was larger in the exudate and the levels were kept throughout the entire course of this inflammation. Arg-Pro-Pro-Gly-Phe ([1-5] BK) level, measured by an EIA newly developed, was much higher than that of des-8,9-BK in the exudate. Intrapleural administration of soy bean trypsin inhibitor (0.3 mg per rat) reduced the levels of both des-8,9-BK and [1-5] BK in the exudates. Reduction in the residual levels of plasma prekallikrein (P-Kall) and high-molecular-weight-kininogen (HMW-K), not low-molecular-weight-kininogen (LMW-K), were accompanied with increase in these BK degradation products, indicating that plasma prekallikrein was activated in the pleural cavity. On the other hand, intravenous injection of acetaldehyde to rats pretreated with disulfiram resulted in the significant increase in the levels of [1-5] BK in the blood, which was accompanied with reduction in the residual levels of LMW-K, not of HMW-K and P-Kall in plasma. These results indicated that the detection of BK degradation products was a good marker for the kinin release in vivo and that the concomitant reduction of the precursor proteins allowed us to identify the type of kallikrein-kinin systems relevant to the kinin release.


Quantification of Ile-Ser-Bradykinin degradation in human serum and ascites.

A leucine aminopeptidase (LAP) and a carboxypeptidase (CP) activity in human serum and malignant ascites degrade Ile-Ser-Bradykinin (ISB = T-Kinin) in two catalytic steps to desArg9Bradykinin. The catalytic activity in serum is always higher than in ascites. In serum the carboxypeptidase activity is higher than the LAP activity whereas in ascites the two activities are not different.


Isolation of a bradykinin-potentiating factor from scorpion Tityus serrulatus venom.

A bradykinin-potentiating factor was isolated and characterized from the scorpion Tityus serrulatus venom by chromatographic techniques and reverse phase followed by biological assays. This factor showed to be able to potentiate the contractile activity of the isolated guinea-pig ileum, inhibited the angiotensin-converting enzyme and potentiated the bradykinin-induced lowering of the arterial blood pressure in the rat.


Bradykinin antagonists: the state of the art.

The availability of practical bradykinin antagonists has revolutionized research in the kinin field. Development of the first antagonists and progress to the present time are reviewed.


Bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers.

A systematic study on dimerization of the bradykinin (BK) antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Leu8-Arg9 has been performed. Several of the dimeric BK antagonists displayed remarkable activities and long durations of action. Rank order of antagonist potency as a function of dimerization position is as follows: rat uterus 6 > 5 > 0 > 2 > 1 > 3 >> 4,7,8,9; guinea pig ileum 6 > 5 > 3 > 2 > 1 > 0 >> 4,7,8,9. These results suggest that the development of BK antagonists of significant therapeutic potential may be possible using a dimerization strategy that can overcome the heretofore limiting problems of potency and in vivo duration of action.


Pseudopeptide analogs of bradykinin and bradykinin antagonists.

Over 100 reduced-bond analogs of bradykinin and bradykinin antagonists were designed, synthesized, and assayed in the classic smooth muscle and blood pressure assays in a further study of the structure-activity relationships of bradykinin. Both potent BK-like agonists and antagonists of BK activity were found among the reduced-bond analogs.


Bradykinin antagonists do not require a D-aromatic amino acid residue at position 7.

More than 200 new analogs of bradykinin have been synthesized and assayed. Analogs containing a D-aliphatic amino acid residue at position 7 and an L-aliphatic residue at position 8 are very potent antagonists of BK action on classical systems. Thus the "conventional wisdom" that a D-aromatic amino acid residue is required at sequence position 7 of BK antagonists must be modified.


Metabolism and characterisation of kinins and Hoe 140 (kinin antagonist) in the synovial fluid of patients with inflammatory joint diseases.

Methods have been optimised for the collection of synovial fluid and the chromatographic separation of individual kinins (bradykinin and kallidin) in the fluid by HPLC. In addition, the stability of the kinin antagonist, Hoe 140, in synovial fluid was compared with that of synthetic bradykinin. Although bradykinin was completely degraded after incubation for only 6 h in pooled synovial fluid obtained from patients with rheumatoid arthritis, Hoe 140 was stable for as long as 2 weeks under the same conditions. These studies will provide quantitative information regarding levels of kinins in inflamed joints and an insight into the therapeutic potential of kinin antagonists.


Immunovisualisation of plasma prekallikrein and H-kininogen on human neutrophils and in human hepatocytes.

Both plasma prekallikrein and H-kininogen were immunolocalised in human hepatocytes by the use of immunocytochemical techniques in conjunction with the confocal optical scanning microscopy. In contrast, both proteins were demonstrated on the external surface of human blood neutrophils. However, detection of H-kininogen on non-fixed but not on fixed neutrophils with the anti-domain 6 antibody (directed at the prekallikrein binding site on H-kininogen), suggested that access to the epitope was blocked by the presence of the bound plasma prekallikrein. Therefore, we prepose that H-kininogen provides the binding site for plasma prekallikrein on circulating neutrophils.


Hormonal regulation of pituitary glandular kallikrein: a morphometric study.

We have previously identified the lactotrophs as the Glandular Kallikrein (GK) containing cells in the rat anterior pituitary using immunocytochemistry, this localization has been independently confirmed with similar methods by other groups. The purpose of the present work was to evaluate the estrogen and dopaminergic control of the GK-containing cells using a morphometric analysis. Female (200-250 g) ovariectomized rats (n = 40) were treated with estradiol (5, 10, 50 micrograms/rat) in the presence or absence of haloperidol (2.5 mg/Kg). The pituitaries were fixed by perfusion with Bouin's and immunostained for prolactin (PRL) or GK. The number of cells and the intensity of the staining were determined by morphometric analysis. Little GK staining was observed in pituitaries from ovariectomized rats, whereas estradiol treatment produced a marked increase in GK staining; GK-positive lactotrophs increased from 4% in control to 75% with 5 micrograms of estradiol, higher doses produced little further increase. However, GK staining intensity in lactotrophs was markedly dependent upon estradiol dose increasing 4-fold between 5 micrograms and 50 micrograms. Haloperidol (2.5 mg/Kg) elicited weak GK staining in 46% of the lactotrophs in the absence of estradiol, and potentiated GK staining intensity elicited with low doses of estradiol. Estradiol also produced a dose-dependent increase in pituitary mass and % lactotrophs indicating lactotroph proliferation. Estradiol produced a dose dependent increase in pituitary wet weight, % PRL-positive cells and % GK-positive cells. Pituitary weight was correlated with % lactotrophs (r = 0.992), and % GK cells (r = 0.874), and % lactotrophs was correlated with % GK cells (r = 0.978).(ABSTRACT TRUNCATED AT 250 WORDS)


Kallikrein-producing cells in the rat pituitary and pineal gland.

By using an immunohistochemical technique, kallikrein-producing cells in the anterior pituitary of rats were identified to be the same as prolactin-producing cells. Kallikrein was localized at the Golgi apparatus, the rough endoplasmic reticulum and secretory granules. Kallikrein was also located in the perivascular cells of the pineal gland.


Cellular localization of human kininogens.

An immunocytochemical screening using polyclonal and monoclonal antikininogen antibodies was performed in various human tissues including blood cells. By comparing the spatial relationship between the cellular localizations of tissue kallikrein and kininogens it was evident that in some tissues both enzyme and substrate were present establishing a close anatomical relationship whereas in others only one of the components could be detected. This pattern of distribution suggests that within various tissues (cells) the major function of either tissue kallikrein (kininogenase, processing enzyme) or kininogen (kinin precursor, cysteine protease inhibitor, kallikrein acceptor molecule) could be different and probably specific to each cell type.


Expression of human salivary-gland kallikrein in insect cells by a baculovirus vector.

A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.


Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.

Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.


Kinetics of bond cleavages at kallidin release by tissue kallikrein: cleavage of two peptide bonds in a single enzyme-substrate complex?

The kinetics of the release of kallidin, L- and KL-chains from bovine L-kininogen by porcine tissue kallikrein were followed and individual kinetic constants for cleavage of the Met-360 and the Arg-370 bond determined. The results suggest that both these bonds in L-kininogen r are hydrolyzed "simultaneously" without appearance of a free singly-nicked intermediate. Kallidin release in the human analogous system is also compatible with such a mechanism.


Kinetics of Lys-bradykinin release by porcine pancreatic kallikrein from rabbit low molecular weight kininogen.

The initial rates of Lys-bradykinin release by porcine pancreatic kallikrein from rabbit low molecular weight kininogen are found to follow the Michaelis-Menten kinetics with kc = 0.62 sec-1 and Km = 1.93 microM at substrate concentrations 0.3-1.3 microM, but at higher ones the Michaelis dependence is broken. Inhibition of the reaction by its product(s) with Kp < Km is revealed with integral analysis methods in a range of 4.5-270 microM kininogen.


Characterization of kallikrein isolated from rat submandibular glands by a simple and rapid purification procedure.

Numerous biochemical properties (e.g. Mr, carbohydrate content, pI) were determined for kallikrein isolated from rat submandibular glands by a simple, rapid purification procedure. The kinetic behaviour of the enzyme towards various inhibitors and synthetic substrates was investigated. The effects of different salts and detergents on the esterolytic activity of the rat tissue kallikrein were recorded.


Role of triglycerides in coronary artery disease: lessons from the Prospective Cardiovascular Münster Study.

The incidence of atherosclerotic coronary artery disease (CAD) was assessed in 4,576 male participants of the Prospective Cardiovascular Münster (PROCAM) study, aged 40-64 years, over a 4-year follow-up period. In this time, 122 study participants developed atherosclerotic CAD (89 definite nonfatal myocardial infarctions and 33 definite atherosclerotic CAD deaths). Univariate analysis revealed a significant association between the incidence of atherosclerotic CAD, and high-density lipoprotein (HDL) cholesterol (p < 0.001) and triglyceride (p < 0.01) levels. The relation to HDL cholesterol remained after adjustment for other risk factors. By contrast, the relation between the incidence of atherosclerotic CAD and triglycerides disappeared if, in a multivariate analysis by means of a multiple logistic function, cholesterol or HDL cholesterol was taken into account. However, the data suggested that hypertriglyceridemia is a powerful additional coronary risk factor, when excessive triglycerides coincide with a high ratio (> 5.0) of plasma low-density lipoprotein (LDL) cholesterol to HDL cholesterol. Even though the prevalence of this subgroup was only 3.7%, it included a quarter of all atherosclerotic CAD events observed.


Triglyceride levels and the risk of coronary artery disease: a view from Australia.

A prospective study of cardiovascular disease in elderly Australians commenced in 1988 in Dubbo, New South Wales. The study population comprised 1,237 men and 1,568 women aged > or = 60 years. The prevalence rates of coronary artery disease (CAD) and putative risk factors were examined cross-sectionally in the baseline data. The age-standardized rate of CAD was 23.8/100 men and 18.1/100 women. In a univariate analysis, the major risk factors for CAD were hypertension, diabetes, family history, reduced high-density lipoprotein (HDL) cholesterol levels, and increased triglyceride levels. The prevalence rate of CAD was examined in those with low-density lipoprotein (LDL):HDL ratios < 5.0 or > 5.0. Most notably in women, the CAD rate was 16/100 with an LDL.HDL ratio < or = 5.0 and 28/100 with an LDL.HDL ratio > 5.0. In the latter group, the rate was 21/100 in those with triglycerides < or = 2.3 mmol/liter and 36/100 in those with triglycerides > 2.3 mmol/liter. In a multiple logistic model that controlled for many potential risk factors or confounding variables, CAD in men was significantly predicted by age, hypertension (odds ratio = 1.40), family history (odds ratio = 2.05), and low HDL cholesterol (odds ratio = 0.78). Significant predictors in women were age, years of education (odds ratio = 0.82), hypertension (odds ratio = 1.45), family history (odds ratio = 1.77), serum triglycerides (odds ratio = 1.30), and low HDL cholesterol (odds ratio = 0.73). An independent gradient of CAD risk with increasing triglyceride levels and a similar gradient with decreasing HDL cholesterol levels were found in women.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypertriglyceridemia: risks and perspectives.

The evidence linking hypertriglyceridemia and coronary artery disease (CAD) is reviewed. A positive correlation between plasma triglyceride level and CAD incidence has been demonstrated in most prospective studies on univariate analysis. However, the significance is weakened on multivariate analysis, in particular when level of high-density lipoprotein (HDL) cholesterol is taken into account, perhaps because of the close metabolic interrelation between the triglyceride-rich lipoproteins and HDL particles. Recent analyses of clinical data have shown that the combination of elevations of low-density lipoprotein cholesterol and triglyceride and low levels of HDL cholesterol confers particularly high risk for CAD. The U.S. National Institutes of Health Consensus Development Conference on Triglyceride, High Density Lipoprotein, and Coronary Heart Disease in February 1992 made recommendations to integrate more fully HDL cholesterol and triglyceride levels into the assessment and treatment of dyslipidemia and CAD risk. Treatment of hypertriglyceridemia should focus on diet and weight control, exercise, and smoking cessation, as well as control of other major risk factors for CAD, notably hypercholesterolemia and hypertension.