Loss of glomerular polyanion correlated with albuminuria in experimental cadmium nephropathy. The relationship between proteinuria and glomerular polyanion (GPA) charge has been studied in a model of experimental cadmium (Cd) nephropathy. Female Sprague-Dawley rats were administered Cd in drinking water for up to 18 months. From month 2, the animals showed an elevation of albuminuria preceding by about 6 months the rise of urinary beta 2-microglobulin and IgG. The nephrotoxic action of Cd was not readily detectable on the basis of the urinary output of beta-N-acetylglucosaminidase, alanine aminopeptidase and lactate dehydrogenase. These enzymes showed either little variation or were affected late in the intoxication process. Administration of Cd for 12 or 18 months did not impair the GFR. The glomerular origin of the albuminuria induced by Cd was demonstrated by estimating the glomerular filtration of rat or human (injected intravenously) albumin in rats whose tubular reabsorption had been blocked by a saturating dose of cytochrome C. The GPA charge was assessed by measuring the binding of the cationic dye, Alcian blue (AB), to membranes of isolated glomeruli. The sialic and sulfate content of these membranes was also determined. The Cd induced-albuminuria was negatively correlated (r = -0.73; n = 37) with the AB binding to glomerular membranes, their sialic acid content (r = -0.39) but not with their sulfate content (r = -0.15). A negative correlation (r = -0.62; n = 37) was also observed between the albuminuria and red blood cell membrane negative charges largely contributed by sialic acid. All these observations can be interpreted as the evidence that Cd enhances the glomerular filtration of proteins through a GPA depletion involving mainly sialic acid.


Effects of exogenous factors on the cerebral glutathione in rodents. Since glutathione is thought to be involved in cerebral functions, changes in the glutathione level imply modulations of the neurotransmission in addition to all the known effects of GSH. It was investigated whether alterations of the cerebral glutathione can be induced by consumption of GSH, by inhibition or stimulation of the synthesis of GSH, or by an inhibition of the re-reduction of the oxidized glutathione. Aminophenazone, propyphenazone, acetaminophen, phenytoin, morphine and nitrofurantoin, known to deplete hepatic GSH, had no effects on cerebral GSH. Diethyl maleate (0.6 ml/kg) decreased the cerebral content of GSH and GSSG in adult rats as well as in fetuses. The depletion of the cerebral GSH caused by diethyl maleate treatment for 4 days was followed by an increase up to 125% and a subsequent return to the normal level after 1 week. In rats starved up to 71 h deficiency of exogenous amino acids caused only a minimal or no decrease in cerebral GSH. The specific inhibitor of the gamma-glutamylcysteine synthetase BSO only depleted GSH in the brain of young mice following the repeated s.c. administration of a high dose (890 mg/kg). After cobaltous chloride (20 mg/kg; twice a day for 2 or 4 days) the GSH level in the brain was unchanged. In vivo inhibition of the cerebral glutathione reductase was caused by ammonium metavanadate (12.5 mg/kg; three times a week for 6 weeks). Nitrofurantoin (150 mg/kg) had no effect. After lomustine (10 mg/kg) a minimal increase in glutathione reductase was found, but simultaneously also an increase in GSSG and of the ratio GSSG/total glutathione.


Effect of ten thiocompounds on rat liver DNA damage induced by a small dose of N-nitrosodimethylamine. The use in a chemoprevention study of high doses of the genotoxic agent might result in erroneous information because of possible nonlinearity of pharmacokinetic processes and toxicity-induced derangement of physiological defense mechanisms. According to these premises ten thiocompounds, potentially active as inhibitors of metabolic activation and/or scavengers, were examined for their capability of reducing the frequency of liver DNA lesions induced by a very small dose of N-nitrosodimethylamine (NDMA). This was accomplished by means of a viscometric technique previously found suitable to detect a minimal amount of DNA fragmentation. Rats were injected i.p. or i.v. with 1 mmol/kg of thiocompound, 0.2 mg/kg NDMA given by gavage 1 h afterwards, and killed for DNA damage assessment 14 h later. Statistically significant changes of viscometric parameters, which are considered indicative of a protective activity, were produced by disulfiram (DSF), and to a lower extent by diethyldithiocarbamate (DEDTC). Any modification of NDMA-induced DNA damage was absent in rats pretreated with glutathione reduced form (GSH) and dimethyl sulfoxide (DMSO). Allyl disulfide (ADS), L-cysteine (CYS), N-acetylcysteine (NAC), alpha-mercaptopropionylglycine (MPG), ethylxanthic acid (PEX), and 2-mercaptoethane sulfonic acid (MESNA) increased in various degree the frequency of DNA-strand breaks. In subsequent experiments the protective activity of DSF was found to be dose-related, dependent on the time of administration, and greater by oral route. Taken as a whole, these results suggest that several putative anticarcinogens might be ineffective against the DNA-damage produced by the low doses encountered in human exposure.


Experimental data from closed chamber gas uptake studies in rodents suggest lower uptake rate of chemical than calculated from literature values on alveolar ventilation. Experimental data obtained in vivo with the closed-chamber gas uptake technique have been reported for a series of volatile chemicals. Pharmacokinetic analyses of these data have been performed either by using a two-compartment model or physiological models. In the former the transfer rate of chemical from ambient air to body is defined by the clearance of uptake. In the latter models the transfer rate depends on alveolar ventilation, cardiac output, and blood: air partition coefficient. In this communication we describe the quantitative relationship between clearance of uptake and alveolar ventilation, cardiac output, and blood: air partition coefficient. Theoretical values of clearance of uptake were calculated for a variety of volatile chemicals using literature data on alveolar ventilation, cardiac output, and blood: air partition coefficient. For most chemicals the experimentally determined values in rats and mice were about 60% of the theoretical values. This suggests that the inhalatory uptake rate of chemical may be overestimated if literature values of alveolar ventilation are used in physiological pharmacokinetic models for rodents.


Accidental death caused by the absorption of 2,4-dichlorophenol through the skin. A case involving an accidental fatality resulting from skin absorption of 2,4-dichlorophenol in a factory is presented. The compound was quantified using gas chromatography after separation on a SP-1240 DA column and detection with flame ionization. Confirmation was obtained with mass spectrometry. Blood concentration was 24.3 mg/l. Other drugs, including ethanol, were not detected.


Histogenetic aspects of the composition and structure of human ectopic enamel, studied by scanning electron microscopy. This study was made on 12 enamel projections and 12 enamel pearls on human permanent molars to compare their structure and composition, which might elucidate possible histogenetic differences between these two forms of ectopic enamel. The enamel projections contained approx. 2 (weight)% less Ca and P than control occlusal and mid-coronal enamel. Their mineralization was less homogeneous and probably defective. Their enamel structure was markedly irregular in the prismatic as well as the aprismatic layer, but the composition and structure were very similar to those of the control enamel close to cervical border. The enamel of the pearls largely corresponded in composition and structure to the control occlusal and mid-coronal enamel. The enamel of large pearls also resembled coronal enamel in its compositional and structural variability in the occlusal-cervical direction. These findings suggest that amelogenesis in enamel projections is a continuation of amelogenesis in the cervical region. In contrast, amelogenesis in enamel pearls follows the same pattern as that in a dental crown, from the occlusal region to the cervical enamel border. Therefore, the enamel pearl may well be regarded as an attempt at new tooth formation.


The influence of morphological variation on Candida albicans adhesion to denture acrylic in vitro. Using denture acrylic pieces coated with either whole human stimulated saliva or oral streptococci, the binding ability of three different Candida albicans strains was investigated. The C. albicans strains include a clinical isolate with the commonly observed, smooth, round colonial morphology (strain 613p), a morphological variant spontaneously derived from the clinical isolate strain 613p (strain 613m1BK) and a clinical isolate from an oral lesion that was also a morphological variant upon primary isolation (strain 228). Levels of adhesion to the acrylic pieces were determined radiometrically using C. albicans cells metabolically labelled with [35S]-methionine. Whole stimulated saliva significantly increased the binding of all strains compared to uncoated acrylic. However, the level of binding of strain 613p to saliva-coated acrylic was significantly greater than the levels observed for the morphological variant strain 613m1BK. Coating acrylic pieces with either Streptococcus sanguis NCTC 10904, Strep. mutans GS-5 or Strep. sobrinus ATCC 27352 instead of saliva resulted in significantly greater binding by strain 613p compared to uncoated acrylic. Pre-coating the acrylic with the oral streptococci did not significantly increase the binding of morphological variant strains 613m1BK and 228 compared to uncoated acrylic. In general, preincubation of adherent streptococci with sucrose to induce the synthesis of extracellular carbohydrate polymers did not significantly increase the binding levels of the C. albicans strains above those observed using streptococci in buffer alone. Compared to its parental strain 613p, morphological variant strain 613m1BK adhered poorly to denture acrylic coated with either salivary constituents or oral streptococci, while strain 228 adhered to the same substrates at an intermediate level. Furthermore, physical disaggregation of clusters of the morphological variant strain 613m1BK did not appear to increase its binding capacity to saliva-coated denture acrylic. The effect of whole stimulated saliva on the adherence of C. albicans 613p to a variety of plastic substrates in addition to denture acrylic was examined. Overall, saliva pre-coating of the various plastics promoted C. albicans 613p adhesion. The adhesion of strain 613p to denture acrylic coated with whole stimulated saliva from each of five different donors or with parotid and submandibular/sublingual saliva from each of two donors was also examined. Regardless of donor, a coating of whole stimulated saliva significantly increased the binding of strain 613p to denture acrylic compared to uncoated acrylic. In addition, a coating of parotid saliva significantly increased the binding of strain 613p to denture acrylic compared to submandibular/sublingual saliva.


The correlation of temporal regulation of glycosaminoglycan synthesis with morphogenetic events in mouse tooth development. The purpose of this study was to investigate the pattern of sulphated glycosaminoglycan synthesis during morphogenesis and cytodifferentiation in mouse tooth rudiments and to compare the results with those obtained in another study for salivary gland, a branched organ. Sulphated glycosaminoglycan was labelled by incubating molar rudiments from day 15 of gestation to day 1 post partum in medium containing [35S]-sodium sulphate. The rudiments were washed, homogenized and digested in pronase and then were sequentially digested by chondroitinase ABC and chemically degraded by nitrous acid oxidation. The fractions from each of these procedures were analysed by chromatography on Sephadex G-50 columns. The analysis revealed that, during morphogenesis, levels of chondroitin sulphate increased to a peak of 91% at day 18 and levels of heparan sulphate diminished to 8% during this period. As cytodifferentiation occurred, the level of chondroitin sulphate dropped to 64% and that of heparan sulphate increased to 35%. These results are similar to those reported for rat submaxillary gland, a branching organ. It appears that this pattern of sulphated glycosaminoglycan synthesis is not a unique feature of branching morphogenesis but may be one which marks the transition between morphogenesis and cytodifferentiation in non-branching rudiments as well.


The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption. The toothless (tl) rat is an osteopetrotic mutation characterized by a generalized skeletal sclerosis, reduced bone resorption, few osteoclasts and a total absence of erupted teeth. This mutation is not cured by bone marrow transplants from normal littermates. It is known that the skeletal defects in tl rats are greatly improved after treatment with colony-stimulating factor-1 (CSF-1). This investigation concerns the effects of CSF-1 on the development and eruption of the dentition of tl rats. Untreated tl rats had no erupted teeth by 56 days after birth, and the roots of incisors and molars were severely distorted by compression against bone. The apex of the mandibular incisor did not extend past the first molar and continued growth of its apical end produced odontoma-like masses consisting of distorted dentine and enamel matrices. In addition, few osteoclasts were seen on alveolar bone surfaces surrounding the developing teeth. Mutants given CSF-1 were characterized by delayed eruption of all molars and sometimes incisors. The incidence of incisor eruption was related inversely to the age at which CSF-1 treatment began. Molars of treated tl rats had well-developed roots similar to those in normal rats. Treated mutants had numerous osteoclasts in alveolar bone and well-developed haemopoietic marrow spaces in the mandible. Histochemical staining for both tartrate-resistant acid phosphatase and tartrate-resistant acid ATPase was reduced or negligible in osteoclasts of untreated tl rats, heavy in normal osteoclasts and of intermediate intensity in CSF-1-treated mutants.(ABSTRACT TRUNCATED AT 250 WORDS)


Increase of microliths in inactive salivary glands of cat. Secretory inactivity could be a factor in the formation of microliths, and so their occurrence in feline salivary glands after the secretory inactivity produced by parasympathectomy was investigated. Parasympathectomy was followed by a greatly increased occurrence of microliths in the submandibular salivary gland, but not in the parotid and sublingual, which may relate to residual secretory activity in these glands. This discovery suggests that secretory inactivity may indeed be a factor in the production of microliths in human salivary glands, and consequently of chronic sialadenitis and sialothiasis.


Transforming growth factor-beta 1 mRNA in neonatal ovine molars visualized by in situ hybridization: potential role for the stratum intermedium. Human dentine contains relatively large amounts of transforming growth factor-beta (TGF-beta), which might originate from odontoblasts. The expression of the TGF-beta 1 message in developing teeth was examined by in situ hybridization. The analysis was made on 5-microns serial sections of mandibular third molars of neonatal sheep cut from tissues that had been fixed in glutaraldehyde and paraffin-embedded. A 35S-labelled cRNA probe, complementary to TGF-beta 1 mRNA, was constructed from human TGF-beta 1 cDNA. Northern analysis of total RNA from sheep placenta and neonatal third molars demonstrated hybridization to a single 2.4 kb TGF-beta 1 transcript from both tissues, indicating cross-reactivity of the human probe in the sheep. In the neonatal molars, in situ hybridization was observed in cells of the inner enamel epithelium, mature ameloblasts and mature odontoblasts, but not within preodontoblasts before dentine matrix formation. TGF-beta 1 mRNA expression was also evident in the cells of the dental papilla but scarcely so in the stellate reticulum. The most striking feature was the appearance of hybridization signal in the cells of the stratum intermedium before hybridization was evident in the inner enamel epithelium. Control sections incubated with RNAase before incubation with probe did not show evidence of hybridization. These findings suggest that TGF-beta 1 may have an important regulatory role in the differentiation of ameloblasts and odontoblasts, perhaps by modulating matrix formation during amelogenesis or odontogenesis. They also suggest a potential novel regulatory role for the cells of the stratum intermedium.


The origin of short- and long-latency mylohyoid nerve responses elicited by high-intensity electrical stimulation of intradental nerve in cats. A double reflex response of the mylohyoid nerve to the jaw depressor muscles after electrical stimulation of the intradental nerves was recorded in cats anaesthetized with alpha-chloralose. The threshold value of the long-latency (16-19 ms) response was almost 20 times greater than that of the short-latency (5-8 ms) response. At the threshold intensity of the long-latency response, the short-latency response with shorter latency appeared. After extirpation of the stimulated tooth pulp, the short-latency response with a lower threshold disappeared, whereas the short-latency response with a higher threshold and the long-latency response both persisted and showed no change in threshold or latency. These findings indicate that the low-threshold, short-latency response was due to excitation of pulpal nerve fibres, while the high-threshold, short-latency response and the long-latency response were both due to excitation of extrapulpal afferent fibres as a result of the spread of current outside the tooth.


The thickness of the sound and periodontally diseased human cementum. Cementum of teeth previously analysed for fluoride was re-examined in order to determine whether or not periodontal disease had affected the thickness of the tissue. In the periodontally diseased teeth the cementum was thinner than normal in the middle region of the root. The apical cementum, however, was significantly thicker in periodontally diseased than in the sound teeth of subjects over 60 yr old. This may account for the higher total fluoride levels previously reported in the apical cementum of these same teeth.


The hearing loss in Wegener's granulomatosis: relationship between hearing loss and serum ANCA. We observed the change of hearing for 1 year in a patient with Wegener's granulomatosis. We also studied anti-human neutrophil cytoplasm antibody (ANCA), which is believed to be specific for this disease and to be associated with disease activity. From these progresses, changes in hearing were considered to be generally related with the general condition and with the ANCA titer level. Because hearing loss in our case was improved by steroids and immunosuppressants, and because ANCA is the autoantibody observed in angiitis syndrome, hearing loss in this case of Wegener's granulomatosis was suspected of being caused by angiitis of the middle and inner ear induced by autoimmune reaction.


Interlay method for myringoplasty.

Interlay method for myringoplasty. Sixty-nine ears with perforation of the tympanic membrane were reviewed. All had undergone myringoplasty by the interlay method. The average period for complete epithelization was 16.1 days after surgery. Neither blunting of the anterior tympanomeatal angle nor lateralization of the tympanic membrane was observed. The success rate was 94.2% for the initial surgery, when "success" is defined as closure of the perforation and complete epithelization (dry ear). The surgical technique and various advantages of the interlay method of myringoplasty are described.


Importance of atticotomy in chronic otitis media with fixation of ossicles. Seventy-seven patients with chronic otitis media were reviewed. Of the 77 patients, 42 underwent tympanoplasty with a columella, and 35 underwent myringoplasty and atticotomy. Twenty-five of the latter 35 patients showed less than 10 dB of hearing improvement with a patch test before operation. The ossicular chain was not disconnected in any of the 25 patients, but the mobility was severely restricted. It was interesting that fixation of the malleus was found at surgery in 80% (20/25) of the patients, whereas the stapes was immobilized in only 16% (4/25). Myringoplasty and atticotomy instead of tympanoplasty with a columella was sufficient to achieve hearing improvement in most of such cases. However, conventional atticotomy was not able to achieve improvement in the mobility of the ossicles because the mobility is usually severely restricted at the malleus. Removal of the anterior tympanic spine is the key to recovery of the mobility of the malleus, resulting in hearing improvement. Rate of success was obtained in 24 of 25 ears (96%).


Four cases of metastatic lymphoma into the temporal bone. A histopathological study on 17 temporal bones obtained from 9 patients who died of malignant lymphoma revealed metastasis of malignant lymphoma in 7 temporal bones from 4 of them. There were no differences in stages at the initial diagnosis between the cases with metastasis and those without metastasis. However, the higher was the frequency of metastasis, the longer became the period from the first medical examination to death. In addition, malignant lymphoma was considered to metastasize into the temporal bone by the following two routes, 1) direct infiltration or invasion from the cerebrospinal fluid and 2) hematogenous metastasis or invasion.


The place of conservation surgery for T3 laryngeal carcinomas with fixation. Although the traditional surgical treatment of T3 laryngeal carcinomas is total laryngectomy, we have obtained favorable survival results for selected cases with partial laryngectomy, as exemplified in the literature. Extending the indications up to ultimate limits by partial, but radical surgical techniques is the recent trend in the world, for the conservation surgery of laryngeal cancers. The primary treatment of T3 laryngeal cancers, instead of irradiation, should be surgical and, for select cases partial laryngectomy, depending on laryngeal embryological development and lymphatic drainage, may be carried out. We have performed partial laryngectomy with elective or therapeutic and radical or modified radical neck dissection for 43 T3 laryngeal carcinomas at the Department of Otolaryngology, Istanbul Medical Faculty, University of Istanbul in the years 1978-1991 and obtained 2, 3, and 5 years of survival rates, which are 89, 79.4, and 73%, respectively.


Surgical treatment of large epiglottic cysts with a side-opened direct laryngoscope and snare. Two cases of large epiglottic cysts are reported. Histopathological examination revealed a retention cyst in one case and an epidermoid cyst in the other. We designed a side-opened direct larngoscope and snare for treating epiglottic cysts. This direct laryngoscope is characterized by an opening from the anterior tip to the posterior end of the right side. The removal of the cysts was accomplished by using this direct laryngoscope and a snare under inhalation anesthesia by fiber optic guided endotracheal intubation. The side-opened direct laryngoscope enabled easy insertion of the snare through the right side opening.


A case of delayed endolymphatic hydrops. A 30-year-old male with delayed endolymphatic hydrops was given a sac operation. An intradural portion of the endolymphatic sac was resected and the epithelial surface was examined under a scanning electron microscope. The epithelium showed fibrous degeneration and disappearance of the epithelial cells. Pathogenesis of degeneration of the endolymphatic sac is discussed.


A case of pharyngolaryngeal stenosis in Behçet's disease. A rare case of pharyngolaryngeal stenosis in Behçet's disease is reported. A 44-year-old female who had Behçet's disease for 13 years complained of dysphagia and dyspnea, and was referred to our hospital for treatment in May 1988. The bilateral meso-hypopharyngeal walls were swollen, and the epiglottis was edematously folded, occluding the larynx. Epiglotectomy and thyrohyoidpexia were performed, with dysphagia and dyspnea disappearing after the operation. Two years after the operation, dysphagia and dyspnea recurred because of two stepped cicatricial stenoses at the mesopharynx. An enlargement operation, incising the pharyngeal cicatricial tissue bilaterally and covering raw surfaces with free skins and the pedunculated cervical flap, was performed. Colchicine, corticosteroid, and tranilast (anti-allergy drug) were administered to prevent re-stenosis. Stenosis was not observed for 10 months after the second operation. Two cases undergone surgical interventions have been reported, and this is the first case reporting long term result of the surgical procedure. Because the skin and mucosa of patients with Behçet's disease shows hyperreactivity to stimuli, the choice of surgical procedure is controversial and recurrence of stenosis is possible.


Effects of prostaglandin E2 on the fluctuating hearing loss in Ménière's disease. To elucidate the possible mediating role of prostaglandins as underlying mechanism of the acute audiologic effects of furosemide in patients with Ménière's disease, the hearing threshold shift after a 1 hour infusion of a commercially available prostaglandin E2 derivative (120 ng/kg body weight) was determined in 10 patients. Prostaglandin E2 produced a transient hearing improvement comparable to the effects obtained in the furosemide test with 1.2 mg/kg bw in the same patients. The effectiveness of a prostaglandin infusion on hearing levels may be a further sign of the involvement of this mediator in hearing processes.


Exercise, oxidative damage and effects of antioxidant manipulation. Exercise induces free radical formation in muscle and liver, and oxidative damage, such as lipid peroxidation. The amount of damage depends on exercise intensity, training state and the tissue examined and can be reduced through dietary supplementation of antioxidants such as vitamin E and possibly coenzyme Q10. Supplementation with antioxidants does not increase maximal aerobic capacity or maximal exercise capacity; effects on endurance capacity are unclear. Deficiency of vitamin E or vitamin C greatly reduces endurance capacity, whereas selenium deficiency has no effect on endurance capacity. In studies by the authors, urinary output of the oxidatively damaged RNA base 8-hydroxyguanosine was not affected by several submaximal exercise bouts nor by supplementation with vitamins E and C and beta-carotene in moderately trained humans. In rats, endurance training caused an increase in oxidative damage, as measured by the protein carbonyl concentration of muscle, but not liver. Muscle protein carbonyl concentration returned to normal on detraining. These results indicate that the search for oxidative damage due to exercise and the effects of antioxidant manipulation on such damage should ideally involve examination of several indices of oxidative damage in various tissues after exercise and training.


Three ways of abolishing automaticity in sinoatrial node: ionic modeling and nonlinear dynamics. A review of the experimental literature reveals that there are essentially three qualitatively different ways in which spontaneous activity in the sinoatrial node can be abolished. We show that these three ways also occur in an ionic model of space-clamped nodal membrane. In one of these three ways, injection of a current pulse abolishes ("annihilates") spontaneous action potential generation. In the other two ways, as some parameter is changed, one sees a sequence of qualitative changes in the behavior of the membrane as it is brought to quiescence. In one of these two ways there are incrementing prepotentials intermixed with action potentials, with a maintained small-amplitude subthreshold oscillation being the limiting case of such behavior. Thus both experimental and modeling work indicate that the number of ways in which spontaneous activity can be abolished, or initiated, in the sinoatrial node is limited. The classification into three ways is based on ideas drawn from the qualitative theory of differential equations, which are introduced. The classification scheme can be extended to encompass behaviors seen in other cardiac oscillators.


Firearm homicide among black teenage males in metropolitan counties. Comparison of death rates in two periods, 1983 through 1985 and 1987 through 1989. To identify US counties (1) that had either significantly high or significantly low firearm homicide rates among black males 15 through 19 years of age in 1983 through 1985 and in 1987 through 1989, and/or (2) that experienced a significant increase in the firearm homicide rate between 1983 through 1985 and 1987 through 1989. Using the Compressed Mortality File, a county-level mortality and population database maintained by the National Center for Health Statistics, Centers for Disease Control, Hyattsville, Md, county-level firearm homicide rates are analyzed. Eighty counties with a population of at least 10,000 black males 15 through 19 years of age in 1987 through 1989. Black males 15 through 19 years of age whose underlying cause of death was classified as firearm homicide (E965.0 through E965.4, or E970) in the ICD-9 (International Statistical Classification of Diseases, Injuries, and Causes of Death, Ninth Revision). County-specific firearm homicide rate. In 1983 through 1985 and in 1987 through 1989, seven and 13 counties, respectively, were identified that had significantly high firearm homicide rates. Firearm homicide rates were significantly high in both time periods in the following counties: Los Angeles, California; Wayne, Michigan; Kings, New York; St Louis City, Missouri; and Baltimore City, Maryland. Firearm homicide rates increased significantly between 1983 through 1985 and 1987 through 1989 in 34 of the 80 counties. Twenty counties had significantly low rates in both time periods. Several counties with low rates in 1983 through 1985 experienced significant increases and by 1987 through 1989 were among those with high rates. Surveillance of firearm homicide rates at the county levels in counties with high and with low rates is a necessary first step in the development of successful violence prevention programs. Those counties where rates are high and increasing are the counties that are in greatest need for intervention strategies. Knowledge of the incidence of nonfatal firearm injuries is also needed.


Effects of running training on the blood glucose and lactate in rats during rest and swimming. The purpose of this study was to examine the effect of physical training on the concentrations of glucose and lactate in the blood of rats during rest and after an acute bout of exercise. We used the following types and periods of training; (i) swimming for 4 weeks, (ii) running for 4 weeks, and (iii) running for 10 weeks. The results clearly show that the resting levels of blood glucose was significantly lower in groups trained by either swimming or running than untrained groups. In addition, after the acute exercise of swimming, animals trained by either running or swimming showed a lower increase in the blood lactate than untrained animals. Furthermore, the increases in the blood glucose after swimming were significantly lower in the group trained by swimming for 4 weeks and by running for 10 weeks than in untrained groups. These results suggest that after physical training by running, animals show an adaptation in the changes in the blood glucose and the blood lactate that are induced by a different type of physical stress, swimming.


Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4. The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans. A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity. Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences. Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.


Clinical application of arterialization of portal vein in living related donor partial liver transplantation. Arterialization of the portal vein was employed during hepatic arterial reconstruction in our first few clinical experiences of partial liver transplantation using liver grafts obtained from living related donors. This procedure reduced the time required for revascularization of the grafts to about 25 min, and could in fact reduce the ischemic phase of the grafts. Repeated practice of the clinical transplantation technique has shortened the time needed to complete vascular reconstruction, eliminating the need for this procedure in most of our subsequent cases. In many clinical cases, however, there may be emergency situations which require vascular reconstruction, resulting in a prolongation of ischemic phase and the deterioration of the cellular viability of the graft. In such situations, arterialization of the portal vein can be a useful way to prevent the prolongation of the ischemic phase and to rescue the graft.


Acute rejection in kidney grafts with delayed onset of graft function. A duplex-Doppler study. Forty-five kidney transplant recipients with delayed onset of diuresis due to acute tubular necrosis (ATN) were examined with duplex ultrasonography (DU). Resistive index (RI) was measured on the 4th post-transplant day. Eleven grafts (24%) developed acute rejection. Mean RI prior to rejection of the 4th postoperative day in these grafts was 0.97 and in the 34 grafts which did not develop rejection mean RI was 0.82. There were 2/26 rejections (8%) in the group of grafts with an initial RI below 0.9 and 9/19 rejections (47%) in the group of grafts with RI of 0.9 or above on the 4th post-transplant day. Six months postoperatively there were 2/26 nonfunctioning grafts in the group with lower initial RI values (less than 0.9) and 6/19 nonfunctioning grafts in the group with higher indices (greater than or equal to 0.9). In nonfunctioning grafts a high initial RI (greater than or equal to 0.9) indicates that these grafts will be prone to developing acute rejection.


Multifactorial analysis of the outcome of 6430 cadaver kidney grafts. A total of 6430 cadaver kidney grafts performed within the network of France-Transplant between 1 January 1978 and 1 January 1989 were analyzed. Each case was examined comprehensively in regard to 12 variables. A multifactorial analysis (Cox regression) was used to determine the degree of association between each covariate and the outcome of the graft. The results were evaluated by calculating relative risks of graft failure for each variable. A total of seven covariates appeared to influence graft survival significantly: the period of transplantation (P = 10(-8)), retransplantations (P = 0.003), age and sex of the donor (P = 0.003 and 0.009 respectively), duration of pretransplant dialysis (P = 0.03), pretransplant sensitization to HLA antigens (P = 0.05), and matching for HLA-A, -B, and -DR loci (P = 0.03). This last parameter has previously been reported as influencing the outcome of the graft in seven out of eight international studies carried out using similar methodology.


Low-temperature fluorometric technique for evaluating the viability of rat liver grafts after simple cold storage. Time-dependent changes in the viability of rat liver graft during cold preservation with Euro-Collins solution were evaluated with NADH fluorometry. Correlation between the fluorometric analysis, 1-week survival rate after liver transplantation, and mitochondrial ATP synthesis activity in the early phase after transplantation was studied. Fluorometric study: Rat livers were preserved at 0 degree-4 degrees C for 0-48 h in Euro-Collins solution and then reperfused for 15 min with oxygenated Krebs-Henseleit solution at 4 degrees C. The amplitude (R x A) between the oxidized and the reduced steady-state NADH fluorometric trace and the velocity (R x V) of the trace were determined to evaluate the mitochondrial respiratory chain. The R x A and R x V remained at levels higher than 90% of control after 6-h preservation, while the R x A of the 9-h preservation group and the R x V of the 12-h preservation group decreased significantly compared with those of the control and the 6-h preservation group. Survival study: a 100% survival rate after transplantation was achieved in the 6-h preservation group, whereas the rates were 18.8% and 0% in the 9- and 12-h preservation groups respectively. These survival rates correlated closely with the time-dependent decrease of the fluorometric parameters. Study of mitochondrial phosphorylative activity and energy charge 3 h after transplantation: With fresh grafts, the decrease in hepatic energy charge after transplantation was reduced to 0.79 from the control value of 0.86 by a 30% increase in mitochondrial ATP synthesis ability.(ABSTRACT TRUNCATED AT 250 WORDS)


Intestinal allograft survival in the rat following pretreatment with donor-specific UV-B-irradiated leukocytes and peritransplant immunosuppression with cyclosporine. Previous studies from our laboratory showed that pretreatment with ultraviolet-B-irradiated donor leukocytes (UV-B DL) combined with brief peritransplant cyclosporine (CyA) resulted in indefinite survival of Wistar/Furth rat cardiac allografts in Lewis recipients. This study was designed to examine the effect of pretransplant UV-B DL with or without peritransplant CyA on orthotopic intestinal allografts in the same rat strain combination. The results showed that while low-dose CyA treatment alone (10 mg/kg i.m. on days 0, +1, and +2) had no effect on intestinal allograft rejection, 20 mg/kg (on days 0, +1, and +2) CyA significantly (P less than or equal to 0.001) prolonged graft survival, with 33% of the hosts surviving indefinitely. The highest dose of CyA (30 mg on days 0, +1, and +2) abrogated rejection, but most transplant recipients succumbed to infection and functional ileus due to a toxic side effect of CyA. Pretreatment with UV-B DL on days -14 and -7 alone did not prolong intestinal allograft survival. Combination of a subtherapeutic CyA dose (20 or 10 mg/kg) given on days 0, +1, and +2 with pretransplant UV-B DL on days -14 and -7 did not alter the survival of intestinal allografts compared to treatment with CyA alone. This suggests that pretreatment with UV-B DL with or without peritransplant administration of CyA has no effect on intestinal allograft survival, in contrast to the effect of such combined treatment on cardiac allograft survival, where indefinite graft survival is observed.(ABSTRACT TRUNCATED AT 250 WORDS)


A chronicle of organ transplant progress in Japan. This study records the progress of organ transplantation in Japan by reviewing significant developments in five areas: actual transplant legislation; landmark cases notably affecting public impressions of organ transplants; efforts to establish brain death criteria; action on the part of the Japan Medical Association; and current attempts to legislate on brain death. The account demonstrates how the notions of both brain death and heart transplantation have met with strong resistance. The first and only heart transplant occurred in 1968. Through its historical emphasis, this report reveals that, although opposition has not impeded transplant research, the determination of death and the idea of heart transplants remain highly controversial due to specific religious, philosophical, and cultural factors.


Coping with the stress of illness.

Coping with the stress of illness. How can we cope better, or help others to do so? The answer to this depends on knowing what an individual is coping with. This, in turn, depends on the appraisal, by the individual concerned, of the significance of what is happening for well-being--in effect, the source of harm, threat, challenge. Cases of myocardial infarction, cancer and chronic pain have some harms, threats, and challenges in common but there are also unique factors in each illness. The patient with a chronic illness is continually appraising his or her symptoms, pains, disease progression with respect to their significance for well-being and survival, and coping accordingly. The paper presents a microanalytic, contextual and process-centred approach to coping which is part of a broad system of thought, emphasizing cognitive appraisal and the person's ongoing relationships with the environment as factors of his or her emotional life. Implications for prevention and treatment of illness in a perspective of health promotion are discussed as well as the need for research to predict long-term outcome from stress and coping.


Emerging forces in cancer care.

Emerging forces in cancer care. The mainstream of scientific research is moving towards increasing recognition of the limits of conventional therapies and cautious optimism about the potential for new biological therapies. This situation has given rise to a social phenomenon among cancer patients. Today, in many parts of the world, a significant minority has chosen to engage actively in the fight for recovery. This minority searches for an intelligent integration of efficacious conventional therapies and complementary therapies involving personal trials of intensive health promotion. These trials cover nutritional, psychological, and immuno-modulating approaches to supporting general health. It appears that 10% among those patients achieve exceptional results, another 10% fail, and 80% become "healthy" cancer patients. While these experiences raise many interesting clinical, scientific and policy questions, they have provided a growing body of evidence that a great deal can be done with behavioural approaches, not in terms of sweeping cures but possibly for some gains in life extension and certainly for great gains in the human experience of being healed (or "becoming whole" with cancer).


Conceptual views on quality of life.

Conceptual views on quality of life. Quality of life can be discussed in many ways. One way is to analyse the empirical quantitative approach to determine whether the research categories are operational, empirically unambiguous, and thematically relevant; progress, in the sense of changing the number of research categories, does not take place through a systematic empirical test but through a modification of the theory of quality of life. The current debate on quality of life has included a conflict between natural science and a humanistic approach. Our view is that quality of life must be defined through the difficult concept of happiness. When cancer threatens happiness, not one but two fundamental crises influence quality of life, which we regard as a key concept of a therapeutic method.


Pain: a kaleidoscope of ideas, concepts and approaches. The following pages contain summaries and short statements on chronic pain providing further material for reflection and debate, namely: a) synopses of recent findings on: the physiology and biochemistry of pain; the contribution of psychoneuroimmunology; b) some views on a sociology of pain; c) analysis of various concepts and approaches which consider pain as: a symptom of disease a form of behaviour a psychosomatic reaction and a social learning process a communication phenomenon a symptom of disintegrated life a disruption in the integrity of the system a signal of broken unity a breach in the wholeness of the individual; d) a holistic approach in pain therapy; e) some unanswered questions.


Canada's framework for health promotion and chronic illness. At the first International Conference for Health Promotion, Canada presented "Achieving Health for All: A Framework for Health Promotion". This document reflects current thinking and proposes a conceptual base for the analysis of contemporary and emerging health issues. The approach is founded on the principles of health promotion developed by the World Health Organization and assimilates the themes which subsequently emerged in the Ottawa Charter. As such, Canada's framework presents a context for discussing chronic illnesses and for identifying opportunities that will lead to an intersectoral course of action. The salient features of this framework (Fig. 1) are briefly discussed.


A ten-point plan for meeting carers' needs. The needs of people requiring extra care--people who are elderly, chronically ill or disabled--are now gaining long overdue recognition. Yet the somewhat different needs of those who look after them at home--known as "carers"--remain largely ignored. Two years ago, representatives of a group of organizations in Britain joined together to commission a project to redress this balance. They were concerned both to set out the range of carers' needs and to issue guidance on the kinds of services necessary to meet them. These documents, prepared following extensive consultation with people working in the field, have now been published (Richardson et al, 1989). This short article provides a brief overview of the issues.


Chronic illness: a media perspective.

Chronic illness: a media perspective. The media can play a major role in creating supportive environments. They influence the man in the street as well as the decision-maker. They help set norms; they contribute to fostering community action. The information they transmit carries prestige. Health workers know this. They realize the power of communication. But seldom do they know how to approach media professionals and to provide them with what they need most: adequate, to-the-point and lively information. This paper provides them with useful guidelines on how to develop effective cooperation with the media and "help the media to help them" transmit the health message and create supportive environments.


Place of residence as a health resort for rheumatic patients. This paper deals with institutional aspects of health promotion in relation to a research programme comprising five projects currently in operation in various regions of the Federal Republic of Germany. The aim of these projects, which are supported at the federal level, is to provide locally based, interdisciplinary care for rheumatic patients throughout the course of the disease. Different organizational means are applied by the different projects to realize these goals. The project described here started in 1985 in Unna, a small town of some 60,000 inhabitants located in the Ruhr area. It involves intensive efforts in health promotion, the aim being to firmly establish self-help activities in everyday life. The following gives an overview of the project, its objective, structure and programme--especially the "cure seminars" organized on an outpatient basis--and some of the difficulties encountered with regard to practical implementation and the long-term provision of health-oriented care. The last part of the paper deals with the research component of the model.


The duo-formula.

The duo-formula. This paper describes research on innovative ways to promote the practice of self-care by people with a chronic complaint. Considerable interest in the subject exists, as confirmed by interviews with patient groups and with a random sample of general practitioners, specialists and community nurses. The answers, however, reflected some uncertainty regarding how to carry out self-care effectively. The study reported here is part of a wider programme of research on the area between conventional health care and self-care, including the development of training programmes for medical professionals and lay people.


Effecting institutional change.

Effecting institutional change. The paper deals with the need for institutional change and behaviour change in the medical care and social security system of the Federal Republic of Germany. The data reported are from the Oldenburg Longitudinal Study (OLS), in which a thousand males suffering from a first myocardial infarction have been surveyed with mailed questionnaires five times in five years. In this study, special emphasis has been given to the influence of social support from the spouse, the medical care system, and workmates on the coping process. Special emphasis has been given as well to institutional factors and organizational problems in the rehabilitation system of general practitioners, rehabilitation clinics and the social security system. The paper concludes that there is a clear need for comprehensive services and other measures that promote self-reliance and self-help activities among patients and their relatives. In the literature on heart disease, "health promotion" is mostly defined as behavioural change on the part of the individual--i.e. patients or members of what is conventionally called a "higher risk group". In this paper, however, I shall concentrate on another aspect which is no less important: the need for institutional change and behavioural change in the medical care and social security systems. This change, I argue, is needed in order to promote the health of patients who survived a first myocardial infarction.


Immunological characterization of the guanine-nucleotide binding proteins Gi and Go in rat islets of Langerhans. Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate Mr of 40,000. Antiserum I1C (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets. Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of Mr 39-40,000. Thus, at least two molecular forms of Go are present in rat islets. Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.


Interleukin-1 beta stimulates growth of adrenocortical cells in primary culture. Using tritiated-thymidine incorporation as a measure of cell growth, interleukin-1 beta stimulated the growth of bovine zona fasciculata/reticularis adrenocortical cells after 72 h in primary culture. Within the range of 10-1000 pg/ml, interleukin-1 beta produced over 40% of angiotensin II-stimulated [3H]thymidine incorporation (P less than 0.005 compared with basal for 10 pg/ml and 1000 pg/ml; P less than 0.05 for 100 pg/ml; two-tailed unpaired Student's t-test). Interleukin-1 beta did not directly stimulate cortisol secretion. By stimulating adrenocortical growth, the increase in interleukin-1 during fever provides a potential mechanism for chronically raising glucocorticoid output. This study is the first demonstration of a long-term effect involving interleukin-1 beta on the adrenal cortex.


Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease. Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.


Stimulation of insulin release by vasopressin in the clonal beta-cell line, HIT-T15: the role of protein kinase C. We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 beta-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (Kd) of 2.5 nM and a total number of binding sites (Bmax) equal to 120 fmol/mg protein. [Arg8]-vasopressin (0.1-100 nM) elicited dose-dependent insulin release from HIT cells by up to 25-fold. This increase was dependent on the presence of extracellular glucose and was blocked by omission of extracellular Ca2+ or addition of verapamil. The stimulation was biphasic; a rapid but short-lived large increase in release was followed by a smaller sustained rise. Vasopressin also evoked a marked, concentration-dependent increase in [Ca2+]i which was also biphasic; an initial spike was followed by a sustained elevation. This increase also required glucose and was blocked by the absence of extracellular Ca2+ or the addition of verapamil. Pretreatment of the cells with TPA overnight to deplete protein kinase C activity did not affect the [Ca2+]i or insulin responses to vasopressin. However, short-term exposure to TPA markedly reduced glucose-induced steady-state [Ca2+]i, despite potentiating glucose-stimulated insulin release sevenfold, and blocked the [Ca2+]i increase induced by vasopressin. These inhibitory effects of TPA were absent in protein kinase C-depleted cells and were prevented by staurosporine. TPA had no significant effect on vasopressin-induced insulin release. Vasopressin did not modify the activity of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)


Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity. Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.


Characterization of an ovine glucocorticoid receptor cDNA and developmental changes in its mRNA levels in the fetal sheep hypothalamus, pituitary gland and adrenal. Fetal sheep tissues possess glucocorticoid receptors (GR), and these change in number during the last two-thirds of gestation. There is, however, no information about developmental changes in tissue GR mRNA levels which might account for alterations in fetal GR content. We have therefore cloned and sequenced a 942 bp GR cDNA from a sheep liver cDNA library, and used it to study the relative abundance of GR mRNA in fetal and neonatal sheep tissues. Analysis of the cDNA revealed a partial sequence of the ovine GR which displayed over 80% identity with residues 143-453 in human GR and 163-472 in rat GR. Furthermore, the first zinc finger motif in these receptors was perfectly conserved among species. The relative abundance of GR mRNA was studied in hypothalami, anterior pituitary glands and adrenals in fetuses at days 60-70, 100-110, 125-130 and at term (approximately 145 days), and in newborn lambs. Total RNA extracts (20 micrograms) were analysed by Northern blot analysis. A single 5.6 kb transcript was detected in all three fetal tissues, and its relative abundance did not change significantly throughout gestation. However, in newborn lambs, levels of GR mRNA increased significantly in the hypothalamus and pituitary gland but decreased to undetectable levels in the adrenal. These tissue-specific changes in the relative abundance of GR mRNA did not correlate with alterations in GR content in fetal tissues, which suggests that the latter may reflect alterations in GR mRNA translation, subsequent modifications and/or GR turnover. In addition, the pattern of developmental changes in GR mRNA content of the adrenal differs from that of the hypothalamus and pituitary gland in neonatal lambs, and indicates that tissue-specific factors may influence GR gene expression in neonatal sheep.


Arachidonic acid-induced insulin secretion from rat islets of Langerhans. There is growing evidence that arachidonic acid (AA) and/or its metabolites may be involved in the control of insulin secretion. We have now investigated the effect of AA on insulin secretion from rat islets, and the possible involvement of protein kinase C (PKC) in this process. Exogenous AA stimulated insulin secretion from intact islets at a substimulatory concentration of glucose (2 mM), but did not further enhance glucose-induced (20mM) insulin secretion. AA-induced insulin secretion was temperature dependent. The secretory responses seen at 37 degrees C were totally abolished by reducing the incubation temperature to less than or equal to 34 degrees C. AA-induced insulin secretion was not dependent upon extracellular Ca2+ and was potentiated by omission of Ca2+ or bovine serum albumin from the media. PKC in rat islets can thus be stimulated by AA, but the stimulation of PKC is not required for AA-induced insulin secretion.


The function of conserved elements in the promoter of the mouse angiotensinogen gene. The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.


Photoaffinity labelled rat androgen-binding protein and human sex hormone steroid-binding protein bind specifically to rat germ cells. A specific receptor with high affinity for rat androgen-binding protein (rABP) was identified in isolated adult rat germ cells and in the corresponding plasma membrane-enriched preparations. Binding was reversible and time-dependent, with maximum relative binding after 40 min at 4 degrees C; it was pH-dependent, with maximum binding at pH 6-8. Unlabelled rABP and human sex steroid-binding protein (hSBP), but not lactotransferrin, serotransferrin, asialofetuin, fetuin or bovine serum albumin, competed with labelled rABP for binding sites on isolated germ cells. Scatchard analysis revealed a single class of binding site with apparent dissociation constant (Kd) values of 0.78 +/- 0.04 nM and 0.97 +/- 0.05 nM in intact germ cells and plasma membrane preparations respectively. A Kd of 1.72 +/- 0.12 nM for hSBP showed that the receptor binding site was effective for both androgen-carrier molecules. Labelled rABP incubated with solubilized germ cell membrane fractions at pH 7 formed a complex excluded from Superose 6B mini-gels; this complex was not formed at pH 3. The receptor complex was also abolished in the presence of a 100-fold excess of either unlabelled rABP or unlabelled hSBP, or in the presence of 20 mM EDTA. These results suggest that the plasma membrane of rat germ cells contains a receptor which selectively binds rABP and hSBP.


Identification of nuclear tri-iodothyronine receptors in Sertoli cells from immature piglet testes. The existence of nuclear tri-iodothyronine (T3) receptors in both Sertoli and Leydig cells isolated from immature piglet testes was investigated. The results demonstrated the presence of high-affinity (Kd = 1.09 +/- 0.25 nM), low-capacity (185 +/- 24 pg T3/mg DNA) binding sites for T3 in nuclei from freshly isolated Sertoli cells. No specific binding for T3 was observed in nuclei isolated from Leydig cells. The localization of specific T3 receptors, which might mediate the onset of thyroid hormone action, at Sertoli cell level confirms that these cells are a target for thyroid hormone and strongly sustain the role of the thyroid in the regulation of testicular functions during postnatal development.


Regulation of transcription factor mRNA accumulation during 3T3-L1 preadipocyte differentiation by tumour necrosis factor-alpha. 3T3-L1 preadipocytes differentiate into cells having the biochemical properties of adipocytes; tumour necrosis factor-alpha (TNF) attenuates this process. Inhibition of differentiation by this cytokine, thought to be mediated at the level of transcription, has been investigated by examining the accumulation of mRNA for six transcription factors and three diversely regulated genes during the first 24 h of the differentiation process. Upon induction of differentiation, a rapid and major accumulation of c-fos and jun-B mRNA, which returned to near basal levels within 4-6 h, was observed. In contrast, c-jun mRNA, although rapidly expressed at the induction of differentiation, remained at relatively constant levels throughout the time-course. Exposure of the cells to 5 nM TNF potentiated the accumulation of all three mRNAs but most significantly that of c-jun (12-fold), which remained elevated for at least 24 h after treatment. In control differentiating cells, krox-20 and fos-B were expressed transiently from 30 min to 2 h, while fra-1 mRNA accumulated over an extended period of 1 to 8 h. Again, TNF enhanced the accumulation of these mRNAs. Accumulation of mRNA for C/EBP, a transcription factor proposed to control the expression of genes involved in the terminally differentiated state, was attenuated after exposure of the cells to TNF. Interleukin-6 (IL-6) mRNA was expressed briefly (30 min to 2 h) and again transiently (at 8 h after induction of differentiation). TNF treatment markedly enhanced accumulation of IL-6 message. We propose that an increased cellular content of one or more transcription factors or the suppression of C/EBP may be responsible for the attenuation of differentiation induced by exposure of the cells to TNF.


Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the masu salmon. Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou. The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8.3-15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions. An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.


Production and characterization of recombinant chicken insulin-like growth factor-I from Escherichia coli. Recombinant chicken insulin-like growth factor-I (cIGF-I) has been produced in Escherichia coli after first modifying a plasmid that coded for a human IGF-I (hIGF-I) fusion protein, in order to introduce codons for the eight amino acid substitutions. The cIGF-I fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography to remove proteinases, refolded and partially purified by reverse-phase high-performance liquid chromatography. The fusion protein was cleaved with hydroxylamine after which cIGF-I was purified to homogeneity by three additional chromatographic steps. Recombinant cIGF-I was equipotent with hIGF-I in cell culture bioassays of protein synthesis and breakdown using rat L6 myoblasts and chick embryo fibroblasts. Binding of radiolabelled cIGF-I and hIGF-I was also equivalent in the two cell lines, as was their binding in ligand blots of chicken, sheep and human plasma. The cross-reactivity of cIGF-I in a polyclonal hIGF-I radioimmunoassay was 60% of that observed with hIGF-I. The availability of recombinant cIGF-I will facilitate investigations into the role of IGF-I in chicken growth and development.


Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges. We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for alpha-amino group. Three disulfide bonds were bridged step by step (A6-A11, A20-B22, and A7-B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.


Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers. The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.


Reexamination of the polymeric distributions of kappa-casein isolated from bovine milk. kappa-Casein the stabilizing protein of the colloidal milk protein complex was purified from bovine skim milk by the method of McKenzie and Wake (Biochim, Biophys. Acta. 47, 240, 1961). The preparations were examined by sodium dodecyl sulfate gel electrophoresis in the presence and absence of a reducing agent. In the presence of a reducing agent, the kappa-casein migrates as a single low molecular weight band. However, in the absence of a reducing agent, a characteristic pattern of aggregates of varying molecular weight was observed with components ranging from monomer to octamer in integer steps. Densitometry of the Coomassie blue stained gels showed an almost equal distribution of components in each band; carbohydrate staining showed preferential location of sugar residues in lower molecular weight components. Treatment with chymosin (rennin) caused a downward shift in apparent molecular weight for each band with no change in the relative intensity of the Coomassie blue stained bands. Similar gel patterns were observed in whole caseins and partially purified kappa-caseins, indicating that this size distribution is a natural disulfide-linked reporter for the distribution of kappa-casein in casein colloids (micelles).


Amino acid sequence of the coelomic C globin from the sea cucumber Caudina (Molpadia) arenicola. The sequence of a globin from a marine invertebrate, the sea cucumber Caudina (Molpadia) arenicola (Echinodermata), is reported. This globin, chain C, is one of four major globins found in coelomic red cells in this organism and is the second to be sequenced. Chain C consists of 157 residues, is amino-terminally acetylated, and has an extended amino-terminal region. This globin shares a 60% sequence identity with the other sequenced C. arenicola globin, D chain (Mauri et al., Biochem. Biophys. Acta 1078, 63-67, 1991), but has a 93.6% identity with a globin from another sea cucumber, Paracaudina chilensis (Suzuki, Biochem. Biophys. Acta, 998, 292-296, 1989).


Carnivora: the primary structure of the major hemoglobin component from adult European lynx (Lynx lynx, Felidae). The complete primary structure of the major hemoglobin component from the adult European lynx (Lynx lynx) is presented. Presence of two hemoglobin components and three chains, beta A, beta B, and alpha, identified by gel electrophoresis. The purification of the globin chains achieved by ion-exchange chromatography. The globin chains were digested with trypsin. The peptide generated were purified by reversed-phase HPLC. Sequencing of the native chains up to 42 cycles and of the tryptic peptides were deduced by Edman degradation in liquid- and gas-phase sequencer. The primary structure established aligned with those of human Hb-A. The comparison of lynx globin chains with other representatives of the Felidae, lion, tiger, jaguar, leopard, and cat revealed high homology.


Specific peptide-bond cleavage by microwave irradiation in weak acid solution. A rapid and selective peptide-bond cleavage in weak acid, induced by microwave irradiation, has been developed. The specific cleavage sites of peptide bonds located only at the carboxyl- and amino-terminal ends of aspartyl residues along the peptide chain. A systematic study including the time course for the cleavage of various aspartyl-containing peptides, the effect of the acidity of the reaction solution on the completeness of peptide-bond cleavage, and the relationship between the power of microwave irradiation and the reaction time of cleavage are studied.


Proteolytic digestion of alpha-lactalbumin: physiological implications. The kinetics of the partial digestion of bovine alpha-lactalbumin (alpha-LA) by trypsin, alpha-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of alpha-LA to trypsin and chymotrypsin at 37 and 5 degrees C decrease in the following order: Ca(II)-alpha-LA greater than Zn(II), Ca(II)-alpha-LA greater than apo-alpha-LA. The HPLC digestion patterns of Ca(II)-alpha-LA and Zn(II), Ca(II)-alpha-LA at 5 and 37 degrees C were similar, while the corresponding digestion patterns for apo-alpha-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-alpha-LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded alpha-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37 degrees C due to Zn(II)-induced shift of the thermal transition of alpha-LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II)-specific site does not cause any drastic changes in the overall structure of alpha-LA. The acidic form of alpha-LA (at pH 2.2 and 37 degrees C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or alpha-chymotrypsin at neutral pH. Complexation of alpha-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to alpha-LA may have physiological significance (e.g., for nutritional transport).


Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: separation of dehydrogenase from esterase activity. Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD+ abolished the burst. The Km for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD+ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested the some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.


Perspectives on toxicological risk--an example: foodborne carcinogenic risk. Epidemiologists estimate that approximately one third of all cancer deaths can be attributed to diet. It is instructive to attempt to apportion this dietary carcinogenic risk to the specific classes of foodstuffs and food additives, pesticides, etc., that are typically regulated. When this is done it is evident that virtually all the calculated risk can be attributed to naturally occurring carcinogens in the diet. This article indicates that both epidemiological data and the simplest kind of risk assessment agree that foodborne carcinogenic risk probably overwhelmingly originates from the food itself and not from additives, pesticides, or contaminants.


Safety evaluation of olestra, a nonabsorbed, fatlike fat replacement. Olestra is the mixture of the hexa-, hepta-, and octa-esters of sucrose with long-chain fatty acids from any edible oil. Its physical properties are comparable to those of triglycerides, but it is not digested by lipolytic enzymes or absorbed and therefore is noncaloric. Technically, it can replace fat in a wide variety of foods and can be used to make cooked, baked, and fried foods lower in fat and calories. A Food Additive Petition is under review by the FDA, which is comprised of results of extensive testing in animals and humans. The major areas of investigation are metabolism and absorption, chronic toxicity, mutagenicity, carcinogenicity, reproductive and developmental toxicity, safety for gastrointestinal tract, nutrition, and the potential for olestra to affect absorption of drugs. This testing involved studies in five different species of animals and over 30 clinical investigations. The results of this research support the safety of olestra for use in foods.


Biotechnology-derived foods from animals. American agriculture is being challenged to produce food in a way that is environmentally sound, that enhances human health, is safe, and whose cost is competitive internationally. The tools of biotechnology offer new techniques for modifying biological traits in a much more directed way than is possible with conventional animal breeding and enables us to ask questions and find answers in a way that was not possible in the past. The new tools facilitate disease diagnosis and prevention, help design foods with improved nutritional characteristics, and increase production efficiency. Full realization of the potential of biotechnology will depend upon our ability to deal with public perceptions of "genetic engineering", support for research, and policy issues such as patenting of animals.


Status of regulatory approval of biotechnology-derived plants and animals. Development of new technology brings with it decisions regarding regulation to ensure safety in technology transfer. Properly formulated and administrated, such regulations can act as a catalyst for the transfer of new technology, not a barrier. Such regulations must be based on sound scientific principles, and are intended to provide protection for the public, engender public acceptance, and foster communication with the various segments of society. To develop this new technology in a safe and orderly manner field testing of the products/organisms of biotechnology, especially genetic engineering, has been permitted by the U.S. Department of Agriculture (USDA). USDA permits certify the environmental safety of field testing of plants and microorganisms developed through the use of material derived from plant pests. Permits are issued based upon analyses of the biology of the organism, the affected environment, and the precautions taken to ensure environmental protection. Licenses for the use of veterinary biologics, including recombinant vaccines, are also issued.


A new approach for assessing the dietary exposure to food additives. By analyzing composite samples of brand name foods, a new method called the Dietary Exposure Assessment Method (DEAM), if found to be feasible, may provide useful estimates of daily intake of food additives for major portions of the U.S. population.


Overview of FDA's Redbook guidelines.

Overview of FDA's Redbook guidelines. As part of its responsibility, the Food and Drug Administration provides guidance to industry and the public concerning the procedures and methods for safety assessment of food and color additives. The FDA published its guidelines in 1982 as the so-called "Redbook" ("Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food"). The Center for Food Safety and Applied Nutrition of the FDA is now in the process of revising and updating the Redbook in light of developments in toxicological testing methods and comments from the scientific community and the public. The effort involves a number of center scientists who have special expertise in various areas of toxicology and food safety assessment. The goal is to make the revised Redbook more useful and practical and to address areas of safety assessment not covered in the 1982 edition. The several papers that follow deal with chapters that will be new to the Redbook.


Revisions to the FDA's Redbook guidelines for toxicity testing: neurotoxicity. In 1982, the Food and Drug Administration issued a publication, known as the Redbook, that described the current toxicological principles used for the safety assessment of regulated food and color additives. However, this document contained only minimum reference to neurotoxicity as a specific toxicological concern and only general mention of the types of data that should be collected to detect and assess adverse changes to the nervous system. The general nature of the toxicological information typically derived from studies based on the original Redbook has had only limited use as a guide for comprehensive assessment of neurotoxic hazard. This limitation is one of the issues being addressed in the current efforts to update the information provided in the Redbook. In the revised Redbook, neurotoxicity, encompassing adverse structural and functional changes to the nervous system, is explicitly identified as an important criterion in the assessment of food chemical safety. The proposed strategy for evaluating neurotoxic hazard has a tiered testing approach. Accordingly, testing would initially involve the identification of chemicals presumptively associated with neurotoxic effects. As appropriate, subsequent testing would be carried out to confirm and delineate the scope of the neurotoxicity, to determine the dose response kinetics, and to define the no-adverse-effect levels.


Testing guidelines for evaluation of the immunotoxic potential of direct food additives. Immunotoxicity testing is a new addition to the safety assessment guidelines for direct food and color additives. The approaches and philosophy for this area of specialized testing are consistent with the case-by-case strategy applied in the regulatory approval process, which is based on structure-activity relationships, preexisting knowledge, and projected exposure estimates. Specialized testing such as immunotoxicity is not part of the basic testing requirements, but would be applied when indicators are positive. Concepts for immunotoxicity testing have evolved, in part, from research for evaluating various testing methods as well as specific study designs. This research, conducted over the last decade, has focused mainly on the rat as the rodent species of choice. The miniature swine was evaluated as a nonrodent model. Testing is defined by type 1 and type 2 tests, which differ in that type 1 tests are performed on the same animals used in the core study design. Sets of type 1 and type 2 tests, with reference to the indicators, define various testing levels. Retrospective testing, expansion of basic testing (such as histopathology and serum chemistry profiles), and alternative study designs, which include satellite groups for evaluation of the functional capacity of the immune system, can be considered in the evaluation of immunotoxic potential.


Evaluating the safety of food and color additives with pharmacokinetic data. Pharmacokinetic studies are designed to quantify, as a function of time, the processes associated with absorption, distribution, metabolism, and excretion of a chemical in experimental animals or in humans. Such studies have played an important role in drug safety evaluation and could be very useful in the safety evaluation of food and color additives. This presentation provides an overview of the potential use of metabolic and pharmacokinetic data in the design and evaluation of toxicological studies and in the assessment of the potential hazard to humans from exposure to food or color additives.


Improvement of oral bioavailability of fenbufen by cyclodextrin complexations. The interactions of fenbufen (FB) with alpha-, beta-, gamma-cyclodextrins (CyDs) were studied in aqueous solution and in solid state. beta-CyD formed water soluble complex with FB in the molar ratio of 1:2 (guest:host). The solid complex of FB with alpha-CyD was obtained in the molar ratio of 1:2 (guest:host), while the same with gamma-CyD was obtained as 1:1 ratio. The dissolution rate and bioavailability of FB were significantly increased by the formation of inclusion complexes (alpha greater than gamma-CyD complex). CyDs had no effect on the metabolic time of FB forming two active metabolites, and the bioavailability of metabolites was also increased by complexation of FB with CyDs. The bitter taste of FB powder was reduced by alpha-CyD complexation. The enhanced bioavailability and reduced bitterness of FB by CyD complexations suggested the possibility of applying FB in smaller doses with fewer side-effects.


Increase in thebaine content of Papaver bracteatum Lindl. after colchicine treatment of seeds. Annual fluctuations in thebaine level of individual plants. From colchicine-treated seeds of Papaver bracteatum Lindl. some poppy plants were obtained that developed capsules richer in thebaine than the controls. The individual poppies were analyzed for capsule thebaine content annually for eight successive years, the results revealing significant year-to-year differences. One of the poppies, X7, (ca 5% thebaine) developed capsules consisting partly of polyploid tissue during the first and second year. This plant was propagated vegetatively to give a series (the X7 series) of new P. bracteatum plants. The capsule thebaine content of these individuals differed markedly the first two years, whereupon the alkaloid production decreased and appeared to level out and reach a value still clearly higher than the controls (mean values 2.3% and 1.3%, respectively). From seeds of four of the thebaine-rich poppies of the X7 series, four new series of P. bracteatum plants were obtained. The capsule thebaine level of these was significantly lower than that of the mother plants.


N-substituted (aminomethyl)benzoate 21-esters of corticosteroids as water-soluble, solution-stable and biolabile prodrugs. Various N-substituted 3- or 4-(aminomethyl)benzoate 21-esters of hydrocortisone, prednisolone and methylprednisolone were synthesized and evaluated as water-soluble prodrug forms, with the aim of developing improved preparations for parenteral or ophthalmic administration. All esters were readily hydrolyzed enzymatically by human plasma. The half lives of hydrolysis in 80% plasma ranged from 8 min to 342 min, the rate being dependent on the structure of the amino group and its position relative to the ester moiety, as well as on the structure of the steroid. The esters showed maximal stability in aqueous solution at pH 3-4. From temperature-accelerated studies of the 3-[(4-methylpiperazin-1-yl)methyl)]benzoate ester of hydrocortisone, the shelf life of an aqueous solution (pH 4.0) of this ester was predicted to be 6 years at 25 degrees C. This estimated shelf life is not reduced by precipitation of the slightly soluble parent drug since the ester was shown to be capable of solubilizing hydrocortisone, possibly by self-micelization.


Prodrugs of thiabendazole with increased water-solubility. The poor peroral absorption of benzimidazole anthelmintics limits their usefulness for the treatment of systemic infections such as alveolar or cystic echinococcosis. The low bioavailability has mainly been attributed to the low aqueous solubility of the benzimidazoles. Using thiabendazole as a model compound the prodrug approach was investigated as a mean to obtain derivatives with improved water-solubilities. Bioreversible derivatization of thiabendazole was performed by N-acylation of the benzimidazole moiety with various chloroformates as well as by N-acyloxymethylation. Both the N-alkoxycarbonyl and the N-acyloxymethyl derivatives were readily hydrolyzed to thiabendazole in human plasma and in rat and pig liver homogenates. The pH-rate profiles for the hydrolysis of the derivatives were determined and the lipophilicity of the compounds was assessed by partition experiments. The water-solubility of the N-alkoxycarbonyl derivatives was up to 12 times higher than that of the parent drug. An N-(4-amino-methylbenzoyl)oxymethyl derivative possessed a 300-fold higher water-solubility. The improved aqueous solubility, adequate lipophilicity and chemical stability combined with a facile enzymatic hydrolysis make such derivatives promising prodrugs for benzimidazole anthelmintics with the aim of improving the peroral bioavailability.


Effect of particle size on ocular permeability of prednisolone acetate in rabbits. The ocular permeability of two sieved fractions of prednisolone acetate with a mean particle size of less than 5 microns and 5-10 microns was studied in rabbits. The results show that there was not significant difference between the corneal uptake of the two fractions of prednisolone acetate. Prednisolone acetate was rapidly taken up by the cornea where it is hydrolyzed rapidly to prednisolone. Only prednisolone was detected in the aqueous humor showing that prednisolone acetate is completely hydrolyzed in the cornea. Both fractions produced similar concentrations of prednisolone in the aqueous humor. For prednisolone acetate, the permeability rate rather than the dissolution rate seems to be the rate-limiting step for corneal transport.


Effect of terminal heat sterilization on the stability of phospholipid-stabilized submicron emulsions. Parenteral emulsions similar in composition to the commercially available fat emulsions were prepared using a Microfluidizer. The relationship of pH and terminal heat sterilization by autoclaving on the stability of phospholipid-stabilized emulsions was evaluated. The stability of these formulations was determined by using droplet size and zeta potential measurements. It was found that the zeta potentials of emulsions stabilized by more purified phospholipid were lower in magnitude compared to those stabilized by less purified phospholipid. Furthermore, acidic emulsions (between pH 5.0 and 7.0) showed an increased droplet size upon autoclaving, whereas the emulsions adjusted to an alkaline pH (pH 8.0 to 10.0) did not show a significant change in their droplet sizes upon autoclaving. The zeta potentials of all the emulsions "increased" (in magnitude) upon autoclaving and achieved values between -40 and -50 mV. The results of our studies could be explained on the basis of hydrolysis of phosphatidylcholine at different pH values, leading to a breakdown of the film surrounding the oil droplet, which results in coalescence and/or cracking of the emulsions.


The alkaloids of the roots of Thalictrum flavum L. The main alkaloid in the roots of Thalictrum flavum is berberine. The alkaloids glaucine, thalicsimidine, thaliglucine, thalidazine, hernandezine and thalfoetidine were also isolated.


Optimization of a matrix tablet formulation using a mixture design. The simplex centroid design was applied to the optimization of a modified release tablet formulation. A base granulation was made with the active ingredient naftidrofuryl. The variables investigated included fractions of the excipients microcrystalline cellulose, lactose and dicalcium phosphate dihydrate. The release rate, crushing strength, friability and weight variation were determined as response parameters. Mathematical models were fitted to the data obtained by the lattice method, described by Scheffé and by means of multiple linear regression. Regression analysis indicated a relatively good fit of the models. On the basis of the regression models, contour plots were constructed. An increase in the amount of dicalcium phosphate caused lower release rate and increased weight variation. An increase in the content of lactose showed lower strength and increased friability, whereas an increase in the amount of microcrystalline cellulose had the opposite effect.


Preparation, purification and characterization of chlorohaemin. Preparation of chlorohaemin (CAS 16009-13-5) is performed on the basis of the method of Labbe and Nishida (acetone-acetic acid-SrCl2 method) with some significant modifications. Instead of blood as starting material, a fresh preparation of purified human erythrocytes is used. This avoids any contamination with serum and erythrocyte proteins and lipids of the end product. Special care is taken to remove contaminating globin by introducing some specific purification steps during isolation and recrystallisation. The yield is in the range 65-75% of theory, and the purity of the product is better than 99.9% as shown by elemental analysis and specific tests on various compounds as possible contaminants which originate from the starting material such as lipids and proteins and/or from the different steps of preparation and purification during the procedure. The pure chlorohaemin which is the compound of choice as reference substance for the AHD method in haemoglobinometry is characterized by LSI mass spectrometry (m/z = 616, haemin ion), field desorption-mass spectrometry (m/z = 652), by IR-spectroscopy, and by UV/VIS absorption spectroscopy (pyridine haemochrome spectrum, AHD spectrum).


Incorporation of the enantiomers of lipoic acid into the pyruvate dehydrogenase complex from Escherichia coli in vivo. The uptake of 35S-labelled enantiomers of lipoic acid into cells from Escherichia coli was studied. The R-enantiomer was taken up by a factor of two more efficiently than the S-form. Autoradiography of polyacrylamide gels of partially purified pyruvate dehydrogenase complex from these cells showed that only the R-lipoic acid was covalently incorporated as a cofactor into the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex.


Side chain distribution and enantiomer composition of biodegradable branched polypeptides with polylysine backbone. A detailed investigation is reported about the primary structure of biodegradable branched polypeptides suitable for carrier function. The side chain distribution of poly[Lys(DL-Alam)], m approximately 3.5, the common inside area of a branched polypeptide model system, was determined by automated Edman degradation. These data indicate that 65% of the branches consist of 2-4 Ala residues and 25% of the side chains are longer than 4 amino-acid residues. The enantiomer composition of poly[Lys(Xi-DL-Alam)], (X = Glu, D-Glu, Leu, D-Leu) and of poly[Lys(DL-Ala2.9-Leu0.79)] polypeptides was investigated by HPLC analysis of their hydrolysates following derivatization with N-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The results proved that the synthesis methods applied for the preparation of branched polypeptides produce compounds whose enantiomer compositions are in good agreement with calculated expectations. These findings could be useful for the reliable interpretation of various biological phenomena observed in connection with the presence of L- or D-amino-acid residues in the side chains.


Microbial metabolism of quinoline and related compounds. XIV. Purification and properties of 1H-3-hydroxy-4-oxoquinoline oxygenase, a new extradiol cleavage enzyme from Pseudomonas putida strain 33/1. 1H-3-Hydroxy-4-oxoquinoline oxygenase was purified to apparent homogeneity from Pseudomonas putida strain 33/1 which can use 1H-4-oxoquinoline as sole source of carbon. The molecular mass of the enzyme was determined to 26,000 Da by gel chromatography and by SDS polyacrylamide gel electrophoresis. The enzyme is labile at temperatures above 30 degrees C and has a pH optimum of 8.0. It requires oxygen for the reaction and is significantly inhibited by metal ions like Cu2+, Zn2+, Hg2+ and by 4-chloromercuribenzoate. The enzyme is specific only for 1H-3-Hydroxy-4-oxoquinoline; the apparent Km value for this substrate is 24 microM.