Effects of NIK-247 on CO-induced impairment of passive avoidance in mice. The effect of NIK-247 on carbon monoxide (CO)-induced amnesia were investigated. A step-down type passive avoidance task with mice was used to compare the effects of NIK-247 with those of tacrine. Two types of CO-induced amnesia model, acute and delayed models, were used. The acute amnesia model was developed using mice exposed to CO before memory consolidation, just after training, and a retention test carried out 24 h after training. The delayed amnesia model was prepared 7 days after CO exposure even when the animals were exposed to CO 4 h after training, after memory had consolidated. NIK-247 administered post-training at 0.03-0.3 and 3 mg/kg or pre-retention test (24 h after training) at 0.3 and 10 mg/kg attenuated the acute amnesia. In addition, NIK-247 (0.03, 0.1, 1 and 10 mg/kg) and tacrine (0.03, 0.1 and 1 mg/kg) administered before the retention test (7 days after CO exposure) improved retrieval in the delayed amnesia model. Tacrine (0.01-0.3 and 3 mg/kg), administered post-training, attenuated the acute amnesia but pre-retention test administration did not. The dose-response curves for NIK-247 and tacrine were biphasic bell-shaped. These results indicated that NIK-247 has an improving effect on hypoxia-induced acute and delayed cognitive dysfunction, and suggest that NIK-247 has promise as a nootropic drug for therapy of memory deficits in patients with cerebrovascular-type dementing disorders.


Interleukin-1 alpha-stimulated fibroblast eicosanoid synthesis is not mediated by interleukin-6. Adherent human dermal fibroblasts secreted interleukin-6, prostaglandin E2, prostaglandin I2 and 15-hydroxyeicosatetraenoic acid (assayed by radioimmunoassay) during a 3 h incubation period. Although human dermal fibroblasts did not secrete interleukin-1 alpha or interleukin-1 beta, human recombinant interleukin-1 alpha stimulated arachidonic acid metabolism and interleukin-6 synthesis. This effect was, at least partly, dependent on de novo protein synthesis. In contrast, human recombinant interleukin-6 had no effect on the synthesis and release of the eicosanoids measured. Human recombinant interleukin-1 alpha also stimulated the metabolism of [14C]arachidonic acid, but only if fibroblast were pre-incubated with the cytokine for three hours. Our data indicate that (a) fibroblasts secrete interleukin-6 but not interleukin-1, (b) interleukin-1 alpha, but not interleukin-6, stimulates fibroblast arachidonic acid metabolism and (c) the mechanisms involved in the metabolism of endogenous arachidonic acid are more sensitive to human recombinant interleukin-1 alpha than those involved in metabolism of the exogenous substrate.


Affinity changes in muscarinic acetylcholine receptors in the rat brain following acute immobilization stress: an autoradiographic study. The modifications in rat brain muscarinic acetylcholine receptors induced by acute immobilization stress lasting 10 min or 2 h were analyzed by quantitative in vitro autoradiography. [3H]N-Methylscopolamine ([3H]NMS) was used as a ligand. Immobilization stress for 10 min did not produce any significant change in the properties of [3H]NMS binding sites throughout the brain. In contrast, 2 h immobilization caused a significant increase in receptor affinity (Kd) without modification in the maximal number of receptors (Bmax) in several brain areas such as the caudate-putamen, cortical layers and CA1 field of the hippocampus, among others. These results, found even in animals killed immediately after the end of the immobilization sessions, suggest that immobilization stress induces supersensitivity of muscarinic receptors in certain cholinergic pathways in rat brain.


5-Hydroxytryptamine stimulates 45Ca2+ uptake by human umbilical vein endothelial cells in culture: mediation by 5-HT2 receptor subtypes. In cultured human umbilical vein endothelial cells, 5-HT and alpha-methyl 5-HT stimulated [45Ca2+] uptake in concentration-dependent manner, whereas the 5-HT1 agonists, m-CPP (1-(3-chlorophenyl)piperazine and 2-MPP (1-(2-methoxyphenyl)piperazine), were without effect. In turn, 5-HT-stimulated [45Ca2+] uptake was inhibited in concentration-dependent manners by the 5-HT receptor antagonists ketanserin (5-HT2), LY 53,857 (5-HT2) and methiothepine (5-HT1/2) and to a lesser degeree by MDL 72222 (5-HT3) and BRL 43694 (5-HT3) whereas (+/-)-propranolol (5-HT1) was without effect. These data indicate that 5-HT stimulates Ca2+ uptake by endothelial cells via activation of a 5-HT2 receptor subtype. 5-HT was without effect on de novo prostacyclin (PGI2) synthesis over the concentration of 5-HT that elicited [45Ca2+] uptake. Since 5-HT did not stimulate PGI2, an event associated with an increase in levels of intracellular Ca2+, it is postulated that the uptake of 45Ca2+ reflects changes of Ca2+ at the level of the plasma membrane rather than on intracellular changes. 5-HT-stimulated Ca2+ uptake may be of relevance to endothelium-dependent relaxation, vascular permeability and endothelial repair and proliferation.


Effects of endothelin on intracellular Ca2+ and contractility in single ventricular myocytes from the ferret and human. We investigated the role of endothelin-1 on peak intracellular Ca2+ ([Ca2+]i) and peak shortening of ventricular myocytes (loaded with indo-1/AM) from failing human hearts. 10 nM of ET-1 significantly increased the cell peak shortening (84 +/- 29%, P less than 0.05) without significantly increasing the peak [Ca2+]i (15 +/- 7%, P greater than 0.05). Further studies on ferret cardiac myocytes indicated that in addition to producing dose-dependent (0.1-10 nM) significant increases in peak shortening (max 55 +/- 6% P less than 0.01) and non-significant increases in peak [Ca2+]i (max 35 +/- 19%, P greater than 0.05), endothelin-1 significantly shifted the peak [Ca2+]i-peak shortening curve upward. The results suggest that endothelin-1 acts directly on human and ferret cardiac myocytes to produce a positive inotropic effect that may predominantly be due to an enhanced myofilament Ca2+ responsiveness.


L-687,306: a functionally selective and potent muscarinic M1 receptor agonist. The oxadiazole L-687,306 is a high affinity muscarinic agonist with a N-methylscopolamine/oxotremorine-M binding profile predictive of a partial agonist. L-687,306 showed marked selectivity in functional pharmacological assays. L-687,306 was a partial agonist at muscarinic M1 receptors in the rat ganglion but a high affinity competitive antagonist at guinea-pig cardiac M2 and ileal M3 muscarinic receptors. This compound gives an opportunity to study receptor reserve involved in muscarinic receptors in vitro and in vivo.


Effect of different subtypes of cognition enhancers on long-term potentiation in the rat dentate gyrus in vivo. The effect of four drugs considered as cognition enhancers on the amplitude of the population spikes and on the long-term potentiation (LTP) evoked by perforant path stimulation was investigated in rat dentate gyrus in vivo. LTP was characterized by the absolute increase in the amplitude of the population spikes, which were expressed in mV, contrary to the widely used percentage value, because the absolute increase was independent of the pretetanus level, whereas the percentage increase was found to be negatively correlated with it. Intravenous administration of the drugs (piracetam 500 mg/kg, hydergine 2 mg/kg, vinpocetine 0.1 and 5 mg/kg and physostigmine 0.01 and 0.1 mg/kg) did not influence the amplitude of the population spikes itself. Piracetam and hydergine did not have an effect on LTP, while vinpocetine and physostigmine altered LTP in a similar manner. The higher doses of the two latter drugs, administered 5 min before tetanic stimulation, induced a significant potentiation of LTP, whereas a significant inhibition of LTP was obtained when the drugs were administered 30 min before tetanic stimulation. Based on the results obtained from guinea pig hippocampal slices, an LTP-potentiating effect of all compounds tested could have been anticipated, but this was not supported by our data. The apparent contradiction between the in vivo and in vitro results is discussed.


Effects of bombesin, of a new bombesin agonist (BIM187) and a new antagonist (BIM189) on food intake in rats, in relation to cholecystokinin. To study the mechanism by which bombesin induces satiety, we studied the effect of two new peptides, BIM187, a bombesin agonist, and BIM189, a bombesin antagonist, on food intake in rats fed 6 h a day. BIM187 at 4 micrograms/kg, significantly reduced food intake at 30 min, but did not change the total 6-h food intake. BIM189 (10 mg/kg), had no effect on food intake when administered alone, even at high doses (20 mg/kg). BIM189 selectively reduced bombesin-induced satiety but had no effect on satiety induced by BIM187. To examine the extent to which the satiety effect of bombesin or related peptides depends on the release of cholecystokinin (CCK), we studied the ability of CCK antagonists, BIM18216 and L364718, to reduce satiety induced by bombesin and BIM187. Neither BIM18216 nor L364718 alone had an effect on the 30-min food intake. They were not able to reverse the effect of bombesin on food intake. In our model, bombesin seems to act on satiety by a mechanism independent of CCK.


Tolerance and withdrawal to chronic morphine treatment in the week-old rat pup. Chronic treatment of adult animals with morphine results in tolerance but there are fewer reports on the effects of chronic opiates during ontogeny. The present experiments assessed the development of morphine-induced tolerance and withdrawal in infant rats. Pups were injected with morphine twice daily from ages 1-7 days and then tested on day 7 for morphine-induced analgesia in a hot-water immersion test, and separation-induced ultrasonic vocalizations in response to isolation from the dam and littermates at 7 and 10 days of age. Tolerance occurred to the analgesic effects of morphine but not to its suppression of ultrasonic vocalizations. Separation-induced vocalizations were greatly increased in chronic morphine-treated pups following naltrexone-precipitated withdrawal at 7 days of age. The increase in ultrasonic vocalizations following naltrexone treatment in morphine exposed pups may be a developmentally unique sign of opiate withdrawal.


In rat brain amoxapine enhances dopamine metabolism: pharmacokinetic variations of the effect. When rats were given i.p. amoxapine, the drug was biotransformed to 7-hydroxyamoxapine, but not to 8-hydroxyamoxapine. The maximal concentrations of amoxapine and 7-hydroxyamoxapine in the serum and brain were found 30 min after the single injection, and the concentration of the former in the brain was higher than that of the latter. During the chronic treatment the concentration of amoxapine in the brain was much higher than that of 7-hydroxyamoxapine. A single administration of amoxapine increased the brain levels of dihydroxyphenylacetic acid and homovanillic acid. Their highest levels were observed 6 h after the injection. Repeated administration reduced the increases; chronic treatment caused tolerance to the enhancing effects on dopamine (DA) metabolism. Tolerance was observed in both striatum and hippocampus, but not in frontal cortex and hypothalamus. Single or chronic injection did not appear to change the level of DA in the brain. Amoxapine itself could be chiefly responsible for the enhancement of DA metabolism. In addition, the level of 3-methoxy-4-hydroxyphenylethyleneglycol in brain decreased transiently right after the injection of amoxapine, but the norepinephrine level did not seem to change following single or chronic administration of amoxapine.


The distribution of 5-HT3 recognition sites in the marmoset brain. The distribution of the binding of [3H]GR65630 (0.2 nM) to putative 5-HT3 recognition sites in the brain of the common marmoset was assessed using autoradiography. Specific binding was heterogeneously distributed with the highest densities found in discrete nuclei of the lower medulla (nucleus tractus solitarii, dorsal motor nucleus of the vagus nerve, nucleus of spinal trigeminal nerve tract and the area postrema). In forebrain areas, relatively high binding densities were located in the medial habenula nucleus and the hippocampus (CA3, CA4 and fascia dentata). Low levels of specific binding in the rest of the forebrain hindered quantification.


Analysis of the vasorelaxant action of jatrophone in the isolated aorta of the rat: influence of potassium channel blockers. The mechanism underlying the relaxant response of rat aortic rings to the diterpene jatrophone was investigated. Jatrophone (3 and 10 microM) did not affect acetylcholine-induced endothelium-dependent relaxations, but caused concentration-dependent inhibition of noradrenaline (NA)-induced concentrations in unrubbed, and to a lesser extent, in denuded rings. Jatrophone (30 microM) fully prevented responses to angiotensin II and NA, while responses to KCl (up to 220 mM) were unaffected. In depolarizing medium (KCl 40 mM), jatrophone (3-30 microM) antagonized Ca(2+)-induced contractions in a concentration-dependent and noncompetitive manner, while verapamil (10-100 nM) caused a concentration-dependent, rightward displacement and depression of the Ca2+ concentration-response curve. Jatrophone (1 to 300 microM) concentration dependently relaxed rat aortic rings precontraction with either NA (1 microM) or KCl (80 mM), yielding EC50 s of 11 and 24 microM, respectively. These relaxant responses to jatrophone were unaffected by glibenclamide (1 microM), but the concentration-response curve was displaced to the right (2- to 8-fold) by other K+ channel blockers such as tetraethylammonium (10 and 30 mM), 4-aminopyridine (3 and 10 mM) or procaine (1 and 3 mM). These results indicate that jatrophone relaxes the rat aorta, at least in part, by activating K+ channels distinct from the ATP-sensitive subtype. Since jatrophone, like verapamil, relaxed preparations contracted with KCl and inhibited Ca(2+)-induced contractions in depolarized preparations, this diterpene may also block Ca2+ influx through voltage-sensitive channels. However, additional actions of jatrophone on receptor-operated Ca2+ channels causing Ca2+ efflux and/or release cannot be fully ruled out.


Pressor response to posterior hypothalamic administration of carbachol is mediated by muscarinic M3 receptor. Unilateral microinjection of the acetylcholine receptor agonist carbachol into the posterior hypothalamic nucleus evokes a pressor response in the conscious, freely moving rat. To further localize this response 3.3 or 5.5 nmol of carbachol was microinjected in a volume of 50 nl directly into and outside the region of the posterior hypothalamic nucleus. Administration of carbachol outside the posterior hypothalamic nucleus failed to evoke a change in blood pressure indicating that the carbachol-induced pressor response is mediated from the posterior hypothalamic nucleus. Since posterior hypothalamic administration of atropine completely blocks the carbachol-induced increase in blood pressure and atropine blocks the three pharmacologically identified muscarinic receptor subtypes, methylatropine and progressively more selective muscarinic antagonists were administered into the posterior hypothalamic nucleus prior to 5.5 nmol of carbachol. Microinjection of the M1/M2/M3 muscarinic antagonist methylatropine (0.19-12.5 nmol), the M1/M3 antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine; 0.9-3.6 nmol), the M1 antagonist pirenzepine (9.5-38 nmol), the M2 antagonist methoctramine (5.5-44 nmol), or the M3 antagonist p-F-HHSiD (para-fluoro-hexahydro-sila-difenidol; 2.1-8.3 nmol) inhibited the peak increase in mean arterial pressure and the area under the curve of the change in mean arterial pressure versus time plot in a dose-dependent manner. Log ID50s calculated for the antagonists from the dose-response curves were found to correlate significantly with the log Kis of the antagonists for the muscarinic M3 receptor subtype. These results demonstrate that the increase in mean arterial pressure evoked by microinjection of carbachol into the posterior hypothalamic nucleus is mediated by the muscarinic M3 receptor.


Inositol hexakisphosphate excites rat medullary sympathoexcitatory neurons in vivo. Inositol hexakisphosphate (IP6) was microinjected into the rostral ventrolateral reticular nucleus or iontophoresed onto reticulospinal sympathoexcitatory neurons of the nucleus in anesthetized rats. IP6 (5-500 pmol) induced a dose-dependent potent and prolonged elevation of arterial pressure which was mimicked by microinjection of ethyleneglycol-bis (beta-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA), while myo-inositol was ineffective. Iontophoretic application of IP6 and EGTA excited the medullary sympathoexcitatory neurons but myo-inositol had no effect. The effect of IP6 on firing rate of the sympathoexcitatory neurons was abolished by co-administration of Ca2+ ions onto these neurons. The results indicate that IP6 acts extracellularly to excite the medullary sympathoexcitatory neurons. The excitatory actions of IP6 may depend, at least in part, on a calcium-chelating action. Chelation of extracellular Ca2+ might be a physiological action of IP6.


Pharmacological characterization of inositol 1,4,5-trisphosphate binding sites: relation to Ca2+ release. Two subcellular fractions, one enriched in plasma membranes and the other in endoplasmic reticulum membranes, were obtained from WRK1 cells using a combination of differential centrifugations and Percoll gradient fractionation. Specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites were detected in these two preparations. Endoplasmic reticulum membranes exhibited a binding capacity which was about 5-fold higher than that of plasma membranes. Dose-dependent Ins(1,4,5)P3 binding was determined. Experimental data obtained with endoplasmic reticulum membranes could be adequately fitted with a two-site model (a high-affinity binding site with Kd and Bmax values of 0.7 +/- 0.15 nM and 12.9 +/- 5 fmol/mg protein and a low-affinity binding site with Kd and Bmax values of 44.2 +/- 14.6 nM and 143 +/- 43 fmol/mg protein). Both the high- and low-affinity binding sites were selective for Ins(1,4,5)P3. Besides Ins(1,4,5)P3, Ins(1,3,4,5)P4 also discriminated between the two populations of sites while heparin interacted with the high- and low-affinity binding sites with the same affinity. Ins(1,4,5)P3-induced calcium release from endoplasmic reticulum vesicles was determined by monitoring the calcium concentration in the extravesicular compartment with fura-2. Under experimental conditions where the degradation of Ins(1,4,5)P3 was reduced (incubation at 0 degrees C), a high-affinity Ins(1,4,5)P3-induced calcium release (apparent Kact around 20 nM) could be demonstrated. These results suggest that in WRK1 cells, the endoplasmic reticulum is a major site for Ins(1,4,5)P3 action and that the high-affinity binding sites located on the endoplasmic reticulum membranes may contribute to the physiological regulation of the cytosolic free calcium concentration.


Serotonin-binding proteins in the bovine cerebral cortex: interaction with serotonin and catecholamines. The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [3H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [3H]serotonin binding (Bmax = 120 +/- 12 pmol/mg protein, KD = 0.12 +/- 0.04 microM, n = 3). Binding of [3H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (Ki from 0.1 to 0.3 microM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [3H]dopamine is greatly enhanced by Fe2+, Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component of [3H]dopamine is saturable (Bmax = 279 +/- 64 pmol/mg protein, KD = 0.19 +/- 0.02 microM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57-58 kDa on native polyacrylamide gel electrophoresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.


Agonist-independent inhibition of G protein activation by muscarinic acetylcholine receptor antagonists in cardiac membranes. Agonist activation of muscarinic acetylcholine (mACh) receptors in porcine atrial membranes stimulates binding of the GTP analog, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins. In contrast, atropine as well as several other mACh receptor antagonists reduced the binding of GTP[S] below basal values, both in the absence and presence of the agonist carbachol, by a similar maximal extent (about 25%). Evidence is presented that this inhibitory action of atropine was not due to an antagonism of endogenous acetylcholine. Similar to agonist-induced stimulation, antagonist-induced inhibition of GTP[S] binding required the presence of GDP and Mg2+. On the other hand, addition of salts, e.g. NaCl, amplified agonist but reduced antagonist effects on GTP[S] binding. The data presented suggest that agonist-unliganded mACh receptors interact with and activate G proteins in native cardiac membranes and that antagonist binding induces a conformational change of the receptor, which then either does not interact with G proteins or which prevents GDP release from and subsequent GTP[S] binding to G proteins.


Distalization of maxillary first and second molars simultaneously with repelling magnets. The aim of this study was to analyse the clinical and the dentofacial effects of using repelling SmCo5 magnets for distalization of maxillary first and second molars simultaneously. Ten consecutive patients, aged 12.0-15.6 years, with Class II malocclusion and moderate space deficiency in the upper jaw were orthodontically treated using prefabricated repelling SmCo5 magnets. The magnets were attached buccally in the premolar and first molar area to a fixed orthodontic applicance. When the magnets were activated, the molars could move freely distally. The tooth movements were analysed by measurements on dental casts, lateral photographs of dental casts, and lateral skull radiographs before and after treatment. The mean treatment time was 16.6 weeks and all maxillary molars could be distalized to a Class I relationship. The mean molar crown movement was 4.2 mm, and the maxillary first molars tipped distally and rotated disto-buccally by 8.0 and 8.5 degrees, respectively. The reciprocal forces resulted in a slightly increased inclination of the upper incisors. It was found that simultaneous distalization of first and second maxillary molars with repelling magnets could be an alternative to ordinary orthodontic treatment methods. As the molar distalization was achieved during a relatively short period, occlusal adjustment, including uprighting and derotation of the maxillary molars as well as post-treatment retention, seems recommendable.


Orthodontic magnets: effects on gingival epithelium and alveolar bone in monkeys. The purpose of the present study was to examine soft and hard oral tissues in contact with or close to orthodontic magnets following an extended exposure time. Two male monkeys were used in the experiment. Individual silver splints covering the teeth from 17 to 27 in the upper jaw and 32-42 in the lower jaw were made. Co5Sm magnets were inserted with cold-curing acrylic buccally and lingually in the splints. The monkeys were injected with tetracycline at the start of the experiment and after 4 weeks. The epithelial thickness in buccal and lingual mucosa under the magnets and corresponding sites on the control side were estimated. The intensity of tetracycline fluorescence in the bone under the magnets and corresponding control sites was assessed semiquantitatively. A thinner epithelium compared to the controls and a patchy tetracycline-incorporation in the bone adjacent to orthodontic magnets in contrast to a homogeneous tetracycline fluorescent pattern in the controls were the most conspicuous findings. It cannot be excluded that the magnetic field or corrosion products from the magnetic material influenced vital processes in the epithelium and the bone close to the magnets. However, it cannot be concluded from the present study what cellular processes were affected.


Dental arch development in a set of triplets. A set of male triplets, of whom the two youngest appear to be identical, has been followed and recorded on plaster casts from 1 month to young adulthood. Measurements of tooth size, arch breadth, length, and circumference, as well as arch relationship, were made at five stages of development. There was a general tendency for the identical pair to become more similar after puberty, and less like their dizygotic brother. Attention is drawn to the potential value of a longitudinal study on a sample of sets of similar sex triplets each containing two identical individuals.


Effect of the vertical force component of Class II elastics on the anterior intrusive force of maxillary archwire. Class II elastics are usually employed in the treatment of excessive overbite and overjet with the Begg technique. The effect of Class II elastics on bite opening and the extent of such an effect is uncertain. This article, based on measurements of 30 cases, illustrates that the effect of the vertical component of force from Class II elastics in reducing the intrusive force generated by the anchor bands in the upper archwire is less than previously believed. The position of the circles in the archwire for intermaxillary elastics has the greatest influence on the anterior intrusive force of the upper archwire. The direction of the Class II elastics and the length of the dental arch (a negative correlation) came second, while the force actually exerted by the elastics had the smallest influence. It is, therefore, suggested that the position of the intermaxillary elastic circles should be located according to the differing clinical objectives.


Long-term effects of Herbst treatment in relation to normal growth development: a cephalometric study. The aim of this study was to evaluate the short- and long-term effects of Herbst appliance treatment in relation to normal growth and development. The sample consisted of 32 subjects (16 boys and 16 girls) with a Class II, division 1 malocclusion treated successfully with the Herbst appliance for an average period of 7 months. The patients were reinvestigated 6 months post-treatment when the occlusion had settled and at the end of the growth period, an average of 6.7 years (SD = 1.1 years) after therapy. The control group comprised of 32 subjects (16 boys and 16 girls) with excellent occlusion (Bolton Standards). Sagittal, skeletal, and dental changes occurring during three observation periods were evaluated on lateral cephalograms in centric occlusion. On a long-term basis, Herbst treatment improved the sagittal jaw base relationship, but did not normalize it. The sagittal dental arch relationship, on the other hand, was almost normalized.


The application of computerized tomography (CT) in cases of impacted maxillary canines. A comparison is made between conventional X-rays and computerized tomography (CT) based on eight patients with thirteen impacted upper permanent canines. The use of CT yields information which is much more detailed than that observed on conventional X-rays, such as: pathological resorption on adjacent teeth caused by aberrant canines; the localization of the injury and the position of the canine in relation to one or both incisors. The amount of radiation per exposure is still rather high for the CT (Köster and Ewen, 1986). Further research is necessary in order to reduce this exposure and to determine the appropriate and most effective use of CT in the dental field.


Malocclusion traits and articulatory components of speech. The aim here was to determine the effects of particular types of malocclusion including the effects of combinations of different occlusal anomalies on speech articulation. Occlusal anomalies and articulatory speech disorders, a diagnosis based on errors in place of articulation, were determined for 451 students, mean age 23 years. The present results showed that risk ratios for producing consonants too far anteriorly was greater by 4.5 times for subjects with mesial occlusion, 3.7 times for those with mandibular overjet, 3.4 times for subjects with incisal open bite and 1.7 for those with lateral cross-bite compared to individuals without those occlusal anomalies. This study suggests that incisal open bite alone is rarely associated with articulatory speech disorders, but if present they tend to be mild. Incisal open bite combined with other occlusal anomalies, especially with mesial occlusion, is related to more severe misarticulations of consonants.


Dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? Endothelial cells modulate the function of their underlying smooth muscle. Thus, altered endothelial behavior could be important in the pathogenesis of vascular and lymphatic diseases, including human and animal filariasis. Endothelium-dependent relaxation is depressed in both in vivo canine femoral artery of dogs infected with Dirofilaria immitis and in vitro rat aorta exposed to adult D. immitis. The experiments reported here were designed to determine if filarial cyclooxygenase products could depress endothelium-dependent relaxation in vitro. Pretreatment of the parasites, but not the vascular ring, with either indomethacin or aspirin, prevented filarial-induced depression of relaxation. Analysis of heartworm-conditioned medium by gas chromatography--mass spectrometry revealed two peaks in the biologically active medium that were not present in the control. One peak had a retention time and chromatographic profile characteristic of derivatized PGD2 standard, and the other was not identified. Incubation of the vascular ring with PGD2 mimicked filarial-induced depression of endothelium-dependent relaxation at low, but not high, concentrations of acetylcholine. Thus, filarial PGD2 may be involved in altered endothelium-dependent relaxation seen in heartworm-infected dogs.


Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice. In parallel studies of Schistosoma mansoni infections in male and female CBA/J mice, major sex-related differences are seen in the development of infection and disease. Upon equal subcutaneous exposures to 45 cercariae female mice present a more severe clinical course with consequent higher mortality than male mice. By 12 weeks of infection, more than 80% of female mice die, while less than 20% of infected males succumb to infection. This greater index of mortality is apparently due to the higher susceptibility of female mice to the development of adult worms. Exposed to 45 cercariae, virtually all females develop patent infections, but 8-34% of male mice do not do so. Also, the recovery rate of adult worms per cercariae from female mice is much higher than that from males, indicating that schistosomula are more successful in developing into adult worms in female mice. Additional studies indicate that this dichotomy of schistosomiasis in the sexes is not restricted to mice of the CBA/J strain, but also occurs in C57BL/6 and outbred CF1 strain mice.


Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase. Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M(r) ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M(r) range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti-Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5-10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.


Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages. During its life cycle, the protozoan parasite Leishmania major alternates from an intracellular amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.


Plasmodium falciparum: a simple polymerase chain reaction method for differentiating strains. Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difficulties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 microliters of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA-2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory.


Dirofilaria immitis: proteases produced by third- and fourth-stage larvae. A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.


Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata. Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.


Follow-up of patent and subpatent parasitemias and development of muscular lesions in mice inoculated with very small numbers of Trypanosoma cruzi. A sequential analysis of patent and subpatent parasitemias, mortality, and histopathology during acute Chagas' disease experimentally produced by inoculation of 10 or 100 bloodstream forms of Trypanosoma cruzi Y strain in susceptible mice was carried out. Parasites were searched for comparatively using three different methods: direct counting, Ficoll-MI density flotation, and hemoculture. Ficoll-MI density flotation promptly discriminated with high reproducibility subpatent parasitemic states not detected in the blood samples analyzed by direct counting. Despite the high proportion of supposedly uninfected animals and depending on the postinfection time, the majority of the mice had bloodstream parasites at the subpatent level detected by Ficoll-MI, and all of them had muscular lesions during the acute phase. All Ficoll-MI-negative blood samples from infected mice were also negative by hemoculture. Normal mouse blood purposely contaminated with parasite quantities ranging from 200 to 2000/ml was tested comparatively by density flotation and hemoculture and showed frequencies of reisolation varying from 25 to 100%. Overall, these data showed that inoculum as low as 10 infective forms of Y strain is able to induce acute Chagas' disease in susceptible mice and that a subpatent parasitemic state of 600-1000 forms/ml is a common finding. The use of Ficoll-MI to detect subpatent parasitemia is discussed.


Plasmodium chabaudi: estradiol suppresses acquiring, but not once-acquired immunity. This study investigates the effect of estradiol (E) on self-healing of Plasmodium chabaudi malaria in mice of the inbred strain C57BL/10. Our data show: (1) Female mice and male castrates are capable of self-healing infections when challenged with 10(6) P. chabaudi-infected erythrocytes. Self-healing is completely suppressed after pretreatment of mice with 12 micrograms E injected sc twice a week for 3 weeks. (2) The suppressive effect of E is prevented by the estrogen receptor blockers tamoxifen and clomiphene. (3) The nonsteroidal E-agonist diethylstilbestrol (DES) also suppresses self-healing. This suppressive DES effect is prevented by tamoxifen. (4) In mice immune to P. chabaudi, neither survival rate nor the course of parasitemia is affected by E, even at 10-fold higher E doses. Our data suggest that the immunosuppressive action of E is a specific genomic effect, i.e., E-induced gene products prevent the development of protective immunity against P. chabaudi.


Plasmodium falciparum: release of circumsporozoite protein by sporozoites in the mosquito vector. The release of circumsporozoite (CS) protein by Plasmodium falciparum sporozoites was investigated to identify factors regulating this process within infected Anopheles gambiae mosquitoes. The potential for sporozoites to release CS protein in vitro was not dependent upon their site-specific developmental stage (i.e., mature oocysts, hemolymph, salivary glands), their duration in the vector, or their exposure to mosquito-derived components such as salivary glands or hemolymph. The capacity of sporozoites to release CS protein was depressed by mosquito blood feeding during periods of sporozoite migration to the salivary glands, but the effect was only temporary and those sporozoites already in the glands were not affected. Free CS protein in the salivary glands was present in 93.3% of 45 infective mosquitoes. Sporozoites from these same, individual mosquitoes were also tested in vitro for CS protein release. In both cases, the amount of soluble CS protein increased as a function of sporozoite density but the total amount of CS protein per sporozoite became progressively less with increasing numbers of sporozoites. Further experiments showed that sporozoite contact with increasing amounts of soluble CS protein caused a down-regulation of CS protein release. Thus, a primary factor regulating the production and release of CS protein by sporozoites is their contact with soluble CS protein within the mosquito.


Effects of vitamin B12 on plasma melatonin rhythm in humans: increased light sensitivity phase-advances the circadian clock? Vitamin B12 (methylcobalamin) was administered orally (3 mg/day) to 9 healthy subjects for 4 weeks. Nocturnal melatonin levels after exposure to bright light (ca. 2500 lx) were determined, as well as the levels of plasma melatonin over 24 h. The timing of sleep was also recorded. Vitamin B12 was given blind to the subjects and crossed over with placebo. We found that the 24-h melatonin rhythm was significantly phase-advanced (1.1 h) in the vitamin B12 trial as compared with that in the placebo trial. In addition, the 24-h mean of plasma melatonin level was much lower in the vitamin B12 trial than with the placebo. Furthermore, the nocturnal melatonin levels during bright light exposure were significantly lower in the vitamin B12 trial than with the placebo. On the other hand, vitamin B12 did not affect the timing of sleep. These findings raise the possibility that vitamin B12 phase-advances the human circadian rhythm by increasing the light sensitivity of the circadian clock.


Presence of a partial urea cycle in the leech, Poecilobdella granulosa. Ornithine carbamoyltransferase (OCT) and arginase, but not arginine synthetase (AS), were detected in the body wall and gut tissues of the leech. The activities of these enzymes were not altered by starvation. The high activity of arginase in body wall is probably due to the association of the latter with botryoidal tissue. Hirudineans, which evolved from oligochaete ancestors, appear to have lost the citrulline-arginine segment of the urea cycle due to their ammonotelic mode of nitrogen excretion.


Do taste receptors respond to perturbation of water structure? The pmr spin-spin pulse relaxation times (T2 values) of the L-amino acids are examined in relation to their taste threshold values. There is an inverse trend between T2 value and threshold value with a good correlation for amino acids whose natural pH is close to neutrality. These results may indicate that taste receptors respond to perturbation of water structure.


Gastrointestinal transit and digestibility of maltitol, sucrose and sorbitol in rats: a multicompartmental model and recovery study. Using data obtained with a dye marker and the gavage technique, the kinetics of gastrointestinal transit of different loads of sugar substitutes (maltitol, sorbitol) and sugar (sucrose) in the rat were analysed using a linear multicompartmental model over a range from the realistic to the non-physiologic high, of carbohydrate intake levels and using only a few experimental time points. The model gave detailed insight into intestinal propulsion and gastrocecal transit time. Rate constants of transport between the compartments investigated were determined; they showed characteristics which could be related to the substance and the dosage administered. Analyses of the gastrointestinal content and calculations of the intestinal net water movement showed that the digestibility and absorption of the disaccharide sugar alcohol, maltitol, in the small gut depended inversely on the dose ingested. For all substances tested, caloric availability in the small intestine was calculated. At a physiological low level of maltitol intake, the results also indicated an insignificant calorie-saving effect in comparison to sucrose, an effect based mainly on the slow absorption rate of the maltitol cleavage product sorbitol.


Deciduogenic effects of mediators of the polyphosphatidylinositol pathway in pseudopregnant mice. Intraluminal injections (15 microliters) of either concanavalin A (125 micrograms) or ionophore A 23,187 (0.01 mumol) induced a decidual cell reaction (DCR) in the uterus of day 4.5 pseudopregnant mice. However, when these agents were administered in different combinations with each other or with CaCl2 (15 mumol) and phorbol-12-myristate-13-acetate (1.6 nmol), interacting effects occurred to either enhance or inhibit each of the others' independent deciduogenic capacities. The results suggest that the polyphosphatidylinositol pathway and Ca2+ are involved in the induction of the DCR in mice with complex interactions occurring between the active components of the pathway to modulate the outcome of the transformation process.


Effect of monensin and diabetes on asialoglycoprotein degradation in rat hepatocytes. We have studied the effects of two modulations--streptozotocin-induced diabetes in vivo, and the presence of the carboxylic proton ionophore monensin in vitro--on the degradation of 3H-asialoorosomucoid ligand in isolated rat hepatocytes. The ligand was internalized by means of a synchronous wave procedure. Diabetes was associated with a marked decrease in the amount of total degraded radioactive ligand compared to that in normal cells (3.6% and 37.3% of internalized ligand respectively, at 60 min), together with increased secretion of degradation products into the incubation medium (87% and 46.3% of the total degraded ligand was secreted by diabetic and normal cells, respectively). Monensin induced similar effects in normal cells, but had no apparent effect in diabetic cells.


Carbamazepine inhibits NMDA-induced depolarizations in cortical wedges prepared from DBA/2 mice. There is some doubt as to the mechanism of action of the widely-used anticonvulsant drug, carbamazepine. In cortical wedges prepared from genetically epilepsy-prone DBA/2 mice, carbamazepine at therapeutic concentrations (1-10 microM) markedly reduced the depolarization produced by N-methyl-D-aspartate (NMDA). The NMDA sub-type of glutamate receptor has been implicated in the pathogenesis of epilepsy and the inhibitory action of carbamazepine on this response suggests that the anticonvulsant action of the drug may be due to its blockade of NMDA receptor-mediated events.


Phosphoramidon enhances allatostatin-mediated inhibition of juvenile hormone biosynthesis in the corpora allata of the cockroach, Diploptera punctata. Use of enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach, Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10(-7) M) and AST 4 (10(-8)-10(-7) M) were observed in the presence of phosphoramidon (10(-5) M or greater). No significant increase in inhibition were seen in CA from day 6 mated females with AST 4 (10(-9)-10(-7) M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin content in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.


Development of melatonin rhythm in the pineal gland and eyes of chick embryo. A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 18: 6 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.


A multivariate approach to socio-ecological development and endocrine variance in the spotted hyaena, Crocuta crocuta Erxleben. This investigation of the socio-ecological development and endocrine variance in the spotted hyaena, Crocuta crocuta, illustrates that the complexity of a multivariate endocrine system cannot fully be understood using the concept of a class mean (mu). Any comprehensive investigation of a multivariate endocrine system should also include an analysis of variance (sigma), as it may provide additional insights into the dynamics of an endocrine hypervolume. Mean cortisol concentrations could not differentiate between various social and reproductive categories (MANOVA); however, in males an analysis of variance (principal component analysis) indicated that the contrast between cortisol and androstenedione was the principle axis of variance once the androgen secreting ability had been accounted for. On the other hand, the same contrast was found to be the principal axis of variance in the female sub-sample, suggesting that cortisol may play a significant role in regulating the endocrine dynamics of this species, despite showing little variance in mean values among various social and reproductive categories.


Modification of the skin feeding site by tick saliva mediates virus transmission. A tick vector of Thogoto (THO) virus was shown to secrete a factor in saliva which potentiates the transmission of THO virus to uninfected ticks feeding on an apparently non-viraemic host. The effect of the saliva activated transmission (SAT) factor on the virus occurred at the site of inoculation in the skin and was apparent even when the virus was introduced 3 days after the SAT factor. The results suggest that tick saliva can play an important role in disease transmission by virtue of host modification at the site of feeding.


New bone induction by demineralized bone matrix in immunosuppressed rats. Subcutaneous implantation of demineralized bone matrix (DBM) initiates a sequence of developmental events which culminate in endochondral bone formation. To test the effects of T-cell deficiency on new bone formation, the morphology of DBM-induced bone was examined in rats thymectomized at three weeks of age and in thymectomized or nonthymectomized rats lethally irradiated and reconstituted with syngeneic bone marrow. At 24 days after implantation, bone induction in control rats was appropriate for their age, while thymectomized-irradiated-reconstituted rats and thymectomized rats had significantly more new bone and larger bone marrow space than the controls. In non-thymectomized, irradiated and reconstituted rats, bone induction occurred in only 25% of the animals, compared to 95% in other groups.


Protein synthesis during germination of heterothallic yeast ascospores. Protein synthesis during ascospore germination of the heterothallic Saccharomyces cerevisiae strain AP-3 was investigated. Protein synthesis in the germinating ascospores appeared to begin approximately 20 min following glucose initiation. Since RNA synthesis did not start until approximately 70 min after the onset of germination, strain AP-3 ascospores must contain RNA which is ready for immediate translation. Both trehalase and glyceraldehyde-3-phosphate dehydrogenase activities were found to be affected by the onset of germination. Trehalase activity was found to increase severalfold following 60 min of spore germination but remained relatively constant over the subsequent 120 min examined. Dehydrogenase activity was not detectable in AP-3 ascospores but was measurable in germinating ascospores.


Two-state model of Paramecium bursaria thigmotaxis. A theoretical framework has been developed for analysis of the interaction between Paramecium bursaria and a glass surface. Adhesion to and detachment from a solid substrate were considered in the model as transitions between alternative states in cell behavior: (a) swimming, and (b) motionless (positive thigmotactic) state. According to the model and experimental data, a change in the fraction of swimming cells is described by a negative exponential course. The proposed model allows positive thigmotaxis, generally referred to in the literature simply as thigmotaxis, to be considered as the rate-constant of transition into the motionless state. This approach permits quantitative determination of thigmotaxis, and reveals its dependence on the phase of culture growth and the type of medium surrounding the cells. In the mineral maintenance solution, paramecia from the stationary phase of growth swim more slowly than those in the logarithmic phase of growth, and show enhanced thigmotaxis. However, a general relationship between thigmotaxis and swimming speed was not established.


Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda). Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanameii chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue-long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid-long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids with conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).


Thermal injury and ozone stress affect soybean lipoxygenases expression. The effects of thermal injury (cold and heat shock) and ozone treatment on lipoxygenases 1 (LOX-1) and 2 (LOX-2) of soybean seedlings have been investigated. Cold stress led to a decrease of the specific activities of both isoenzymes, attributable at least in part to a down-regulation of gene expression at the translational level. Both heat shock and ozone treatment enhanced lipoxygenases-specific activities, acting at the level of transcription of the genes. It is proposed that LOX-1 and LOX-2 are involved in the thermotolerance of soybeans and in the precocious aging induced by ozone.


Peptidyl-prolyl cis-trans isomerase from Bacillus subtilis. A prokaryotic enzyme that is highly sensitive to cyclosporin A. Cyclophylins are members of a class of proteins with peptidyl-prolyl cis-trans isomerase activity. These enzymes bind the immunosuppressive agent, cyclosporin A (CsA), which acts as a competitive inhibitor. The peptidyl-prolyl cis-trans isomerase from Bacillus subtilis (PPIase) was purified to homogeneity in a 4-step purification procedure, which resulted in a 100-fold protein purification with a yield of 5%. Coomassie blue-stained SDS-PAGE revealed a single band of about 18 kDa. PPIase activity was determined using synthetic peptides as substrates in a 2-step reaction coupled to chymotrypsin. Treatment of Bacillus subtilis PPIase by CsA revealed an inhibition constant of Ki = 175 nM, which differs from cyclophilin of enterobacteria such as E. coli or Salmonella typhimurium and is in the range of human enzymes.


Influence of amphipathic peptides on the HIV-1 production in persistently infected T lymphoma cells. The effects of several amphipathic peptides on HIV-1 production in persistently infected cells are described. Melittin, a 26 amino acid alpha-helical amphipathic peptide, reduces HIV-1 production dose-dependently, whereas other amphipathic peptides do not. Six melittin derivatives which retain the alpha-helical portion have similar effects as melittin. The reduction of viral infectivity is not due to an effect of melittin on the virus particles but to an intracellular action of the peptide, which is readily taken up into cells, as shown by quantitative ELISA. Western blots of cells from melittin-treated cultures suggest that the processing of the gag/pol precursor is impaired.


The effect of dilauroyl-L-3-phosphatidylcholine on the interaction between cytochrome P-450 1A1 and benzo[a]pyrene. Fluorescence quenching of benzo[a]pyrene (BP) by cytochrome P-450 1A1 was used to probe the effect of the lipid, dilauroyl-L-3-phosphatidylcholine, on this substrate-enzyme interaction. In the presence of lipid, a monoclonal antibody to this P-450 maximally inhibited BP binding at an antibody-to-P-450 ratio of 1:2, corresponding to an antibody crosslinked P-450 complex. The antibody did not inhibit BP binding in the absence of lipid. These results indicate that when P-450 is subjected to the orientational constraints imposed by antibody-mediated crosslinking, the lipid alters the conformation or quaternary structure of the P-450 oligomer in a manner which changes its affinity for BP.


Heat and cold denaturation of beta-lactoglobulin B. The thermal denaturation of bovine beta-lactoglobulin B was investigated by high-sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 20 mM phosphate buffer. The process was found to be a reversible, two-state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low-temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of beta-lactoglobulin B.


Effect of pancreatic type phospholipase A2 on isolated porcine cerebral arteries via its specific binding sites. The addition of porcine pancreatic group I phospholipase A2 (PLA2-I) produced a transient contraction followed by a relaxation in helical strips of porcine cerebral arteries. Its ED50 value (2.3 nM) was almost identical to the Kd value (3.9 nM) calculated from the specific binding of 125I-labeled porcine PLA2-I in cultured porcine cerebral arterial smooth muscle cells. Type-specific action of PLA2s and homologous desensitization strongly implicated the involvement of PLA2-I-specific sites in the response. The transient contraction was abolished by treatment with indomethacin as well as by the removal of endothelium, indicating the dependence of vasoconstrictor prostaglandins synthesized by PLA2-I in endothelium. The PLA2-I-induced relaxation response was also observed in bovine and cat cerebral arteries, thus providing a new aspect of PLA2-I as a vasoactive substance.


Differences in the thermostabilities of barley (1----3,1----4)-beta-glucanases are only partly determined by N-glycosylation. Barley (1----3,1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzyme EII carries 4% by weight carbohydrate and is more stable at elevated temperatures than isoenzyme EI, which has no associated carbohydrate. The relationship between carbohydrate content and thermostability has been investigated by treatment of the two isoenzymes with N-glycopeptidase F (EC 3.5.1.52). Removal of carbohydrate from isoenzyme EII results in a decrease in the enzyme's thermostability, but treatment of isoenzyme EI with the N-glycopeptidase F has no effect. In addition, removal of a single N-glycosylation site in isoenzyme EII (Asn190-Ala-Ser) by site-directed mutagenesis of the corresponding cDNA led to a reduction in thermostability, while the introduction of this site into isoenzyme EI enhanced stability. We conclude that N-glycosylation of Asn190 enhances the stability of isoenzyme EII at elevated temperatures, but that other factors related to their primary structures also contribute to the differences in thermostabilities of the barley (1----3,1----4)-beta-glucanases.


A membrane-associated cysteine protease inhibitor from murine hepatoma. A cysteine protease inhibitor was purified from total membrane fractions of an invasive murine hepatoma, Hepa cl 9. On gel filtration under non-reducing conditions the purified inhibitor was eluted in a single peak of M(r) 10-15 kDa, but resolved as two bands at 14 and 70 kDa on SDS-PAGE under reducing conditions. By isoelectric focusing, the inhibitor ran at an isoelectric point of 4.75. Immunoblotting studies using the enhanced chemiluminescence technique indicated no crossreactivity with monoclonal antibodies to stefin B and cystatin C or with a polyclonal antibody to low M(r) kininogen. In contrast, the 14 kDa and 70 kDa bands both crossreacted with a polyclonal antibody to stefin A, suggesting that the cysteine protease inhibitor associated with Hepa cl 9 membranes may be a modified form of stefin A.


The primitive metazoan Hydra expresses antistasin, a serine protease inhibitor of vertebrate blood coagulation: cDNA cloning, cellular localisation and developmental regulation. We have isolated and characterized cDNAs from Hydra which encode antistasin, a potent inhibitor of factor Xa in the vertebrate blood clotting cascade. Hydra antistasin is expressed in gland cells and represents a major class of transcripts from Hydra's head. Sequence analysis revealed that Hydra antistasin contains 6 internal repeats of a 25-26 amino acid sequence with a highly conserved pattern of 6 cysteine and 2 glycine residues identical to that in leech antistasin. Conservation of antistasin in a lower metazoan provides a potential link between the vertebrate and invertebrate coagulation systems.


Over-expression of the small heat-shock protein, hsp25, inhibits growth of Ehrlich ascites tumor cells. hsp25 is a small, growth-related, mammalian stress protein which is highly accumulated in the stationary phase of Ehrlich ascites tumor in vivo. Ehrlich ascites cells cultivated in vitro under conditions of continuous exponential growth express hsp25 only at a low level. These cells were stably transfected with an eukaryotic expression vector carrying the coding sequence of the small heat-shock protein, hsp25, under control of the murine metallothionein promoter. The resulting cell lines (EAT II6 and EAT II8) exhibit constitutive over-expression of the small heat-shock protein, hsp25, which can be further increased by induction with cadmium. Both cell lines show increased thermoresistance. The in vitro proliferation rate of the transfected cell lines EAT II6 and EAT II8 is significantly decreased depending on the degree of cadmium-regulated over-expression of hsp25. Furthermore, a significant delay in Ehrlich ascites tumor growth in mice using the hsp25 over-expressing cells for primary inoculation could be demonstrated.


Endothelin 1 hydrolysis by rat kidney membranes. Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.


The genetic defect of the original Norwegian lecithin:cholesterol acyltransferase deficiency families. Three of the original Norwegian lecithin:cholesterol acyltransferase (LCAT) deficiency families have been investigated for mutations in the gene for lecithin:cholesterol acyltransferase by DNA sequencing of the exons amplified by the polymerase chain reaction. A single T----A transversion in codon 252 in exon 6 converting Met(ATG) to Lys(AAG) was observed in all homozygotes. In spite of the identical mutation, the disease phenotypes differed in severity. This was not reflected in the expression of LCAT in the heterozygotes.


Cloning and sequence analysis of a cDNA clone from Arabidopsis thaliana homologous to a proteasome alpha subunit from Drosophila. A cDNA clone isolated from an Arabidopsis thaliana cell suspension culture library showed considerable similarities to the proteasome 28 kDa alpha subunit of Drosophila [(1990) Gene 90, 235-241]. The 250 amino acid-long protein encoded by Arabidopsis TAS-g64 clone has important homologies in its primary structure and in the predicted secondary structure with the PROS-28.1 clone from Drosophila. The only divergence observed between the two sequences is for the 20 C-terminal amino acids. This subunit might share important functions in both kingdoms, as revealed by the important conservation between plants and animals. In plant cells it is encoded by a single-copy gene and probably regulated by stress and/of division.


The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin. Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.


Influenza virus A infection induces interleukin-8 gene expression in human airway epithelial cells. To determine the role of the airway epithelial cell in mediating virus-induced inflammation, we infected primary cultures of human airway epithelial cells with human influenza type A/Port Chalmers/72 (H3N2). After two days, the medium was collected for measurement of the chemotactic cytokine interleukin-8 by enzyme-linked immunosorbent assay. The RNA was extracted from the cells for analysis of interleukin-8 mRNA by Northern blot analysis. Interleukin-8 production was more than doubled by viral infection, while interleukin-8 mRNA was increased four-fold. Thus induction of interleukin-8 gene expression in virus-infected airway epithelium may be an important early step leading to virus-induced airway inflammation.


Cell-free translation in reversed micelles. Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.


Carbohydrates in mammalian tryptophanyl-tRNA synthetase. Homogeneous preparations of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) contain monosaccharides (mannose, fucose, galactose, N-acetylglucosamine) as revealed by liquid chromatography. Their content comprises 2.5-3.0% (w/w) of the enzyme composed of two subunits (60 kDa x 2). The same set of sugars was detected in elastase and CNBr-generated fragments (with molecular masses of approx. 40 kDa and 30 kDa, respectively). It is concluded that bovine tryptophanyl-tRNA synthetase, in addition to being a metallo- and phosphoprotein, is also a glycoprotein.


Isolation from Spirulina membranes of two photosystem I-type complexes, one of which contains chlorophyll responsible for the 77 K fluorescence band at 760 nm. Two types of chlorophyll-protein complexes of photosystem I (PSIa, PSIc) have been isolated from the membranes of Spirulina platensis using a Triton X-100 treatment and chromatography on DEAE-Toyopearl. The complexes are equally enriched with P700 (Chl: P700 = 100-110) but show different electrophoretic molecular masses--140 (PSIa) and 320 kDa (PSIc)--and differ in the content of long-wavelength absorbing Chl. PSIa has a typical PSI fluorescence band at 730 nm (F730) as the main band at 77 K, whereas PSIc is responsible for F760, the intensity of which depends on the redox state of P700. PSIc only shows 77 K light-induced variable fluorescence at 760 typical of Spirulina membranes and cells.


Direct RNA polymerase chain reaction for TMV detection in crude cell extracts. A part of the 30,000 bp transport protein gene of tobacco mosaic virus (TMV) RNA was amplified via direct RNA PCR and via traditional reverse transcription followed by cDNA PCR. Both amplified cDNA products were restricted with NcoI or HaeIII endonucleases and identical restriction fragments were produced. Two efficient methods of viral RNA concentration from an infected tobacco leaf extract were used: both 3-3.5 M sodium acetate alone and 3 M LiCl with 4 M urea quantitatively precipitated TMV RNA from the extracts. TMV RNA thus obtained could be readily amplified by direct RNA PCR. These results demonstrate that direct RNA PCR can be applied for the detection or/and analysis of high molecular weight RNA and for diagnosis of viral infections.


Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297. We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.


Studies on antisense inhibition of translation in vitro. Anomalies and re-evaluation. Experiments were carried out to better characterize antisense control of translation. Results in an E. coli system confirmed specific inhibition of poly(U) translation. At low concentrations, certain homopolymers (including poly(rA)) stimulated translation. Oligo(dA(n)) was inhibitory at n less than or equal to 8. Translation of globin mRNA in reticulocyte lysates indicated that ssDNA 15-mers targeted at beta-globin mRNA inhibited both alpha- and beta-globin production. Sequences targeted immediately downstream of the AUG were the least effective in inhibition. These and other anomalies are discussed here in relation to those of others, emphasizing caution in performing antisense experiments.


Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. The NiFe hydrogenase from Azotobacter vinelandii is a membrane-bound alpha beta heterodimer that can oxidize H2 to protons and electrons and thereby provide energy. Genes encoding the alpha and beta subunits, hoxG and hoxK respectively, followed by thirteen contiguous accessory genes potentially involved in H2 oxidation, have been previously sequenced. Mutations in some of these accessory genes give rise to inactive enzyme containing an alpha subunit with decreased electrophoretic mobility. Mass spectral analysis of the subunits demonstrated that the alpha subunit had a molecular weight 1,663 Da less than that predicted from hoxG. Since the N-terminal sequence of the purified alpha subunit matches the sequence predicted from hoxG we suggest this difference is due to removal of the C-terminus of the alpha subunit which may be an important step linked to metal insertion, localization, and formation of active hydrogenase.


Protein conformational changes and myelin solubilization by anion-detergent solutions. The addition of sodium sulfate to a myelin suspension in sodium phosphate buffer at neutral pH, containing octyl glucoside detergent (OG), increases the membrane solubility more than 5-fold by an unknown structural mechanism. FTIR spectroscopy has been applied to investigate anion effects on the conformational structure of myelin proteins. Sulfate and sulfate-phosphate media, but not phosphate alone, induce a great conformational protein disorder. The addition of the detergent to the anion mixture solution prevents the myelin from protein denaturation. The conformational transitions have also been quantified through the amide I region. Explanations of these changes and their connections with myelin solubility are also included.


A selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89), inhibits phosphatidylcholine biosynthesis in HeLa cells. In this study, we report that the potent and selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89) interferes with the incorporation of choline into phosphatidylcholine in HeLa cells. Treatment of cells with 10 microM H-89 for 1 h decreases the phosphatidylcholine biosynthesis by 50%. This inhibition is prevented by simultaneous addition of 10 microM forskolin, while the choline uptake itself is not affected by H-89.


Synthesis of a large peptide mimicking the DNA binding properties of the sex determining protein, SRY. The sex determining protein, SRY, has been recently described as containing a DNA binding motif, also called the SRY box. This 80 amino acid box was synthesized using the continuous flow solid-phase technique. The product was then purified and tested according to such diverse criteria as its intrinsic structure or its biological activity (DNA binding capacity), and compared to the full-length protein. The data indicate that the peptide is relevant for the properties described so far for the protein.


Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract. Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.


Synthesis of active metabolite(s) from 1 alpha-hydroxyvitamin D3 by human monocytic leukemia cells. Synthesis of the biologically active metabolite(s) from 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) was examined in various types of human leukemia cell lines. Untreated monocytoid leukemia cells (U937 and HEL/S) metabolized 1 alpha (OH)D3 to the active metabolite(s), possibly 1 alpha, 24- and/or 1 alpha, 25-dihydroxyvitamin D3, and these cells were efficiently induced to differentiate by treatment with 1 alpha (OH)D3. However, the other types of leukemia cells did not efficiently metabolize it and were not induced to differentiate by 1 alpha (OH)D3. The possible therapeutic advantage of 1 alpha (OH)D3 in the treatment of monocytic leukemia is discussed.


Expression of protein kinase C subspecies in rat retina. An extract of rat retina was subjected to Mono Q followed by chromatography on hydroxyapatite, and the protein kinase C (PKC) subspecies were identified by immunoblot and biochemical analysis. It was found that, although the relative activities assayed with myelin basic protein as a common phosphate acceptor vary greatly with one another, the alpha-, beta I-, beta II-, gamma-, delta-, epsilon-, zeta-, and another structurally unknown PKC subspecies are expressed in this tissue. Thus, the retina is a unique tissue which expresses most of the PKC subspecies so far identified in mammals.


Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.


[11 beta-hydroxysteroid dehydrogenase and steroid receptors]. 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), as its name implies, is the enzyme responsible for the conversion of cortisol to cortisone, and of corticosterone to 11-dehydrocorticosterone. Ulick et al. reported the detailed investigation of a patient with the syndrome of apparent mineralocorticoid excess (AME), who had the stigmata of florid hyperaldosteronism but low normal or suppressed levels of renin and aldosterone. Such patients show marked abnormalities of cortisol metabolism. From a series of studies, the consensus grew that AME reflects the absence, or very low activity, of 11 beta-HSD in the kidney of affected patients. In addition to providing a framework for understanding the pathogenesis of AME, these studies prompted a re-evaluation of other areas of steroid in the kidney. Glycyrrhetinic acid, the active principle of liquorice and carbenoxolone, exerted its mineralocorticoid action not by a direct effect on mineralocorticoid receptors but by inhibiting renal 11 beta-HSD, thus producing a mild, drug-induced form of AME. Recently Monder et al. reported the cloning and expression of rat and human cDNA encoding corticosteroid 11 beta-dehydrogenase. The physiological role of 11 beta-HSD in conferring aldosterone-selectivity on otherwise non-selective type I receptors has been focused using the genetic method in addition to the biological ones.


[Diagnostic imaging of the thyroid].

[Diagnostic imaging of the thyroid]. Among various imaging modalities used for the diagnosis of thyroid diseases, ultrasonography is the most sensitive and economical method to visualize anatomic detail and structural relations. Scintigraphy using 123I or 99mTc gives critical information about thyroid cell function and makes it possible to differentiate the causes of thyrotoxicosis. Tumor scintigraphy using 201T1, 67Ga, 131I-metaiodobenzyl guanidine or 99mTc(V)-dimercaptosuccinic acid is useful for the characterization of nodules. Recently introduced magnetic resonance imaging appears promising in demonstrating pseudocapsules around tumors and capsular invasion.


[A case of pseudohypoparathyroidism (PHP) type II associated with Bartter's syndrome--restoration of phosphaturic response to parathyroid hormone (PTH) by treatment for hypopotassemia]. We report a case of PHP Type II whose phosphaturic response to PTH was restored by treatment for complicated Bartter's syndrome. A 34-year-old woman was admitted to our hospital in July 1990 because of tetanic convulsion. The physical examination showed normal blood pressure (118/62mmHg), round face without shortness of metacarpal bones and positive Trousseau's sign. Although renal function was normal, hypocalcemia (6.5mg/dl) and hyperphosphatemia (4.8mg/dl) in association with high levels of serum PTH (942pg/ml) and 1.25 (OH)2D3 (86pg/ml) were disclosed. Ellsworth-Howard test revealed that there was no increase in the urinary secretion of phosphate despite an increase in urinary cAMP excretion. On the other hand, hypopotassemia (2.5mEq/l) and metabolic alkalosis with high plasma renin activity (22.8ng/ml/hr) and aldosterone concentration (22.7ng/dl) were coexistent. Pressor response to angiotensin II infusion was blunted. Although no glomeruli were obtained by renal biopsy specimen, vacuolar degeneration on proximal tubules were noted. These findings indicated that she had PHP Type II associated with Bartter's syndrome. By administration of potassium (24mEq/day), spironolactone (50mg/day) and only small doses of 1 alpha-hydroxyvitamin D3 (0.5mg/day), serum levels of potassium as well as calcium were normalized and tetanic attacks disappeared. In March 1991, she was re-examined by Ellsworth-Howard test in order to clarify the effects of hypopotassemia on renal tubular response to PTH. Interestingly, phosphaturic response to PTH was restored, and the degree of increase in urinary cAMP excretion was 4 times as high as that on the first admission. These results suggest that hypopotassemia changes the response of renal proximal tubular cells to PTH, particularly such as reabsorption of phosphate and cAMP response, although it is possible that hypocalcemia may contribute to the blunted phosphaturic response to PTH. The mechanism of hypocalcemia seen in this case remains to be elucidated.


[Measurement IGF-I in human blood by immunoenzymometric assay]. Insulin-like growth factor-I (IGF-I) is the growth factor which binds to its specific binding proteins in plasma and mediates most of the actions of growth hormone (GH). In terms of this, measurement of IGF-I concentrations is considered to be important in the diagnosis and treatment of growth abnormalities. In the present report we describe a modified immunoenzymometric assay (IEMA) for IGF-I using monoclonal antibody and peroxidase conjugated polyclonal antibody, which was originally developed by Tamura et al. The minimum detection level was 3ng/ml and good linearity was obtained at a range of 3-50ng/ml. The recovery of added IGF-I was nearly quantitative, and cross reaction with human IGF-II was less than 1%. There was a significant positive correlation between the IGF-I values determined by IEMA and by RIA. We measured the levels of IGF-I by this IEMA in normal subjects and children with short stature. Serum or plasma samples were extracted by acid-ethanol method. In normal children IGF-I level was observed age-dependently. IGF-I values gradually increased and reached peak levels (101.2-473.2ng/ml) in pubertal period and thereafter decreased to adult levels (67.3-329.6ng/ml). In subjects with short stature older than 5 yrs, the percentages falling below the normal mean for chronological age by at least 2SD were 100% for complete GH deficiency (n = 5), 80.0% for partial GH deficiency (n = 15), 25.0% for GH neurosecretory dysfunction (n = 4), 40.0% for non-endocrine short stature (n = 60) and 25.0% for Turner syndrome (n = 4). In children younger than 4 yrs, it may be difficult to use IGF-I for diagnosis of GH deficiency because there was an overlap in IGF-I values with normal children. IEMA for IGF-I is shown to be a reliable method for measurement of IGF-I in blood and is appropriate for diagnosis of growth disorders with short stature in subjects older than 5 yrs.


[The role of insulin on renal sodium handling and sympathetic nerve activity in overweight normotensive subjects]. Recently, it was suggested that the role of hyperinsulinemia on the hypertensive mechanism of essential hypertension might be related to renal sodium handling and sympathetic nerve activity, especially in obese hypertensive patients. However, the interrelationship between insulin, obesity, renal sodium metabolism and sympathetic nerve activity in normotensive subjects (NT) still remains unclear. The present study, therefore, was undertaken to clarify the role of insulin on renal sodium handling and sympatho-adrenal function in overweight NT. The study consisted of 24NT, who were divided into two groups of twelve non-obese (NNT) and twelve obese (ONT) subjects. NNT was categorized as a body mass index (BMI) less than, and ONT as a BMI equal to or more than 25kg/m2. In the early morning, after overnight fasting, all subjects remained in a supine state and were examined for renal clearance test. During the two-hour clearance period, mean arterial pressure (MAP), heart rate (HR), endogenous creatinine clearance(CCr), urinary excretion of sodium (UNaV), fractional excretion of sodium (FENa), plasma immunoreactive insulin (IRI), plasma norepinephrine concentration (pNE), and plasma epinephrine concentration (pE) were determined. Although no significant difference was found in age, MAP, HR, pE, CCr or UNaV between the two groups, a significantly higher IRI (p less than 0.05) and lower FENa (p less than 0.05) were observed in ONT than in NNT. There was no significant correlation between IRI and UNaV, FENa or pE in ONT or in NNT. In addition, no significant correlation was shown between FENa and pNE or pE in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction of phospholipids with proteins and peptides. New advances 1990. 1. The review deals with the recent achievements in the study of the various interactions of phospholipids with proteins and peptides. 2. The interactions are classified according to the hydrophobic, hydrophilic or mixed character of the interactive forces. 3. The effect of the interaction on the structure and biological activity of the interacting molecules is also discussed.


Patterns of sulphation in heparan sulphate: polymorphism based on a common structural theme. HS appears to be a well-organised molecule with a domain structure that is apparently unique amongst the GAG family (Gallagher, 1989). Further refinements in sequence analysis are needed to corroborate the simplified model proposed in Fig. 4. It is still not clear why evolution has favoured a structural motif of widely spaced sulphated domains. Presumably, some advantages must accrue to the organism from this design, and one idea, that we have discussed previously, is that the polysaccharide functions as a "template" for the organisation of structural proteins in the ECM and for the binding and presentation of growth factors within the matrix polymer network. The sulphated regions are likely to display considerable conformational versatility as a result of the presence of the iduronate residues, and this property may be very important for the protein-binding properties of the polysaccharides (Casu et al., 1988). Sulphation patterns within these regions could favour oligosaccharide conformations necessary for specific protein interactions. An important question in this context is why different cells express on their surfaces HS with subtle differences in sulphation pattern. Perhaps the polymorphic features of HS are involved in higher-order tissue- and organ-specific mechanisms controlling cellular recognition and morphogenesis. The consistency with which aberrant sulphation of HS is detected in malignant disease (Gallagher and Lyon, 1989) in which cellular recognition and differentiation are impaired, adds some substance to this view.


Glutathione depletion and rabbit renal medulla 6-ketoPGF1 alpha and TxB2: levels in vivo and following homogenate incubation in vitro. 1. The effect of glutathione (GSH) depletion on rabbit renal medullary homogenate 6-ketoPGF1 alpha and TxB2 synthesizing capability was investigated. 2. GSH depletion in vivo with diethyl maleate (DEM) produced higher (P less than 0.05) 6-ketoPGF1 alpha and TxB2 renal medullary levels compared to controls. Homogenization and incubation lowered (P less than 0.05) GSH such that there were no differences in GSH between treatments after 5 min of incubation. By 30 min, GSH was lower (P less than 0.05) and 6-ketoPGF1 alpha higher (P less than 0.05) in homogenates from controls in comparison to those from DEM-treated rabbits. 3. The results indicate GSH depletion increased 6-ketoPGF1 alpha levels in rabbit renal medulla in vivo but subsequent GSH catabolism prevented assessing the effect of this GSH depletion on prostanoid synthesizing capability.


Use of mitochondrial inner membrane proteins and phospholipids to facilitate disengagement of the catalytic and malonyl-CoA binding components of carnitine palmitoyltransferase from liver mitochondrial outer membranes. 1. It was shown by Ghadiminejad and Saggerson (1991) that the anionic detergent cholate caused disengagement of the malonyl-CoA binding entity from the catalytic entity of outer membrane carnitine palmitoyltransferase (CPT1). 2. This disengagement was only observed if inner membrane material was present. 3. It is now shown that this effect is mimicked by a CPT-free inner membrane protein fraction together with an inner membrane lipid extract or with individual phospholipids (phosphatidylcholine, phosphatidylethanolamine or diphosphatidylglycerol). 4. The lipids alone have no effect but act synergistically with the inner membrane protein fraction.


Analysis of the conformation and stability of human bronchial lysozyme by circular dichroism. 1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.


Characterization of polyclonal antibodies against ovine adipocyte plasma membranes. 1. Antisera against ovine adipocyte plasma membranes were developed in a mare. 2. These antisera showed a high degree of specificity to adipocyte plasma membranes and cross-reacted with other tissues. 3. Antisera cross-reactivity can be removed by adsorption of the antiserum with various tissue plasma membranes without significant reduction in their reactivity to adipocyte plasma membranes. 4. Antisera reacted with different affinity to adipocyte plasma membranes from different sites and from different species of animals.


Stabilization of porcine spleen cathepsin A with chaotropic ions and its destabilization with thiols at intralysosomal pH. 1. A correct assay for cathepsin A was developed by adding 0.1 M NaNO3 as an enzyme stabilizer to the assay system. 2. Cathepsin A was purified homogeneously from porcine spleen by DE 52 column, Sephadex G-150 column and Try-Phe-CH-Sepharose column chromatography. 3. The optimum pH of the cathepsin A activity was 4.9, which is near the value of the intralysosomal pH. 4. Chaotropic agents exerted stabilizing effects on the purified cathepsin A activity at pH 5.0, with sodium nitrate being the most effective among the agents tested. 5. Cathepsin A was inactivated rapidly and irreversibly by thiols.


Polyoxyethylene 9-lauryl ether-solubilized alkaline phosphatase: synergistic stimulation by zinc and magnesium ions. 1. Polidocanol-solubilized apoalkaline phosphatase could be stimulated either by zinc ions (Kd = 8.5 nM) or by magnesium ions alone (Kd = 3.8 microM). 2. Zinc and magnesium ions had synergistic effects on Polidocanol-solubilized apoalkaline phosphatase, leading to a fully active enzyme (700-800 U/mg). 3. Zinc ions inhibited non-competitively the Polidocanol-solubilized apoenzyme (Ki = 7.1 microM) by displacing magnesium ions from their binding sites. 4. A model for the action of zinc and magnesium ions on the modulation of the enzyme activity is proposed.


Investigation of the presence of branched-chain alpha-keto acid dehydrogenase in mammalian hepatic peroxisomes. 1. Rat liver was fractionated into peroxisomes and mitochondria and branched-chain keto acid (BCKA) dehydrogenase activity was measured. 2. All BCKA dehydrogenase activity was associated with the mitochondrial fraction and none with the peroxisomal fraction. 3. BCKA dehydrogenase was also not detected in hepatic peroxisomes of rats treated with clofibrate which induces several peroxisomal enzymes. 4. Hepatic peroxisomes from rabbit, hamster and dog also did not show any BCKA dehydrogenase activity. 5. We conclude that mammalian hepatic peroxisomes do not contain BCKA dehydrogenase.