RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content. An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.


Analysis of antibodies and other large glycoproteins in the mass range of 150,000-200,000 Da by electrospray ionization mass spectrometry. The analytical applicability of electrospray ionization mass spectrometry (ESIMS) to large glycoproteins in the molecular weight (MW) range of 150,000-200,000 was demonstrated. Multiply charged ions (charge state as high as 150+) of several typical macrosized glycoproteins of immunological significance were generated by pneumatically-assisted electrospray (ionspray) and their masses measured on a quadrupole mass spectrometer having a mass-to-charge (m/z) range of 2400. The resolution of the quadrupole instrument was insufficient to resolve the glycocomposition microheterogeneities in the MW range studied. Nevertheless, the average MWs of three immunoglobulin G (IgG) class murine monoclonal antibodies, anti-(human alpha 1-antitrypsin) (148,484 +/- 4), anti-(human alpha 1-acid glycoprotein) (149,599 +/- 12) and anti-(beta-galactosidase) (component I, 150,544 +/- 10, and component II, 151,496 +/- 17), and human alpha 2-macroglobulin monomer (186,100 +/- 100), and human complement component C4 (196,863 +/- 29) were still determined from the fused peak profiles of their constituent glyco components (the errors given reflect the measurement precisions of the simultaneous multichannel MW determinations). The difference between the measured average MW and the unmodified sequence MW was used to assess the degree of post-transitional modification in human alpha 2-macroglobulin (13.6%) and human complement component C4 (5.3%). For the large glycoproteins studied here, glycosylation did not appear to seriously affect the effectiveness of the electrospray ionization; up to 70% of their full charge-retaining capacities were fulfilled under the usual experimental conditions. These results show that ESIMS is capable of providing analytically useful information for macrosized proteins.


Trabecular generation de novo. A morphological and immunohistochemical study of primary ossification in the human femoral anlagen. An understanding of trabecular formation in early skeletal development may provide insight into the problem of trabecular replacement in the aging skeleton. In an optical and scanning electron microscope study of the processes of de novo trabecular generation, the immunohistochemical distribution of collagen Types I, II and III, together with the matrix organising proteins fibronectin and tenascin, has been examined in the ossifying human femoral anlage. In the region of the developing spongiosa, the primary osseous trabeculae that arose by endochondral ossification were assembled around calcified cartilage remnants, consisting almost entirely of aggregates of mineralised microspheres. These structures were specifically recognised by antibodies raised against collagen Type II and fibronectin. In contrast, the primary osseous trabeculae that arose by subperiosteal intramembranous processes, were assembled around a framework of prominent coarse fibres that were recognised by antibodies raised against collagen Type III and tenascin. Irrespective of their origin, all the new trabeculae were similar in their general staining character for collagen Type I and fibronectin. However, throughout the developmental stages examined here endochondral trabeculae were separated from intramembranous trabeculae by a discrete boundary of compressed cells and mineralised cartilage.


The effects of growth factors on the day 13 chorioallantoic membrane (CAM): a study of VEGF165 and PDGF-BB. The in vivo effects of two growth factors, VEGF165 and PDGF-BB, were studied in the chick chorioallantoic membrane (CAM). The factors were air-dried on Thermanox discs and the inverted discs were placed on the day-13 CAM for a period of 3 days. The specimens were then fixed, examined under a stereomicroscope and processed for semi- and ultrathin sectioning. VEGF165 induces marked vascular growth. Many new blood vessels emerge from the precapillary arterioles, and a brush-like formation of vessels can be seen in this region. In the venous part of the vascular system, the formation of sinusoidal or lacunar vessels can be seen. Edema does not develop. PDGF-BB induces thickening of the CAM due to extracellular-matrix production and the proliferation or immigration of fibrocytes. These lie just beneath the ectodermal epithelium and are oriented parallel to it. Out of the four factors we have already studied (PDGF-BB, VEGF165, Angiogenin, bFGF), only VEGF165 specifically induces the growth of blood vessels.


Cytokeratin expression and early lens development. Immunohistochemical analysis of cytokeratins and vimentin in human, rabbit and rat lens epithelium during development revealed transient coexpression of both types of intermediate filaments. Cytokeratins were still detectable after the closure of the lens vesicle (rat and rabbit embryos 13 days post conception) and in the epithelial cells located at the anterior side of the lens in 7-week-old human embryos. Different monoclonal antibodies against cytokeratin 8 reacted differently in lens cells but not in other embryonic tissues. In addition, early human and rabbit specimens exhibited cytokeratin immunostaining in the neuroectodermal cells of the eye cup as well as in the surrounding mesenchyme, and in the hyaloid artery. Possible explanations for the loss of cytokeratins during the differentiation of ectodermal and neuroectodermal cells are discussed.


HNK-1 expression pattern in normal and bis-diamine induced malformed developing rat heart: three dimensional reconstruction analysis using computer graphics. The spatiotemporal distribution of the immunoreactivity of monoclonal antibody HNK-1 was investigated immunohistochemically in normal and bis-diamine-induced malformed rat embryonic hearts using three-dimensional reconstruction with computer graphics. First recognized in the primitive heart 11.5 days after conception, HNK-1 immunoreactivity was distributed in the atrio-ventricular and bulbo-ventricular junctional areas with incomplete ring-like appearance in the early embryonic stages. In the late embryonic stages the immunoreactive sites were rearranged and localized in the sites topographically corresponding to almost the entire pathway of the conduction system, including the three major internodal tracts connecting the right sinoatrial node and atrioventricular node. Immunoreactivity gradually decreased after the completion of the conduction system, and only a faint reactivity in the atrio-ventricular node region remained in the new-born heart. These results indicate that HNK-1 is expressed temporarily in the pathways corresponding to the conduction system during the development of the heart. In bis-(dichloroacethyl)-octamethylen-diamine (bis-diamine)-induced malformed hearts, localization of HNK-1 immunoreactivity was not remarkably altered in the early embryonic heart. In the late embryo, immunoreactive sites in the sino-atrial node region and atrio-ventricular node region deviated dorsocaudally with the poorly developed internodal tracts, and abnormal distribution was observed in the bilateral atria. We consider that these abnormalities may occur in conjunction with abnormal morphological development such as insufficient absorption of the sinus venosus.


A comparative study on the effects of tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo. The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax carriers on the CAM: Tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominantly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation.


Migration and distribution of circumpharyngeal crest cells in the chick embryo. Formation of the circumpharyngeal ridge and E/C8+ crest cells in the vertebrate head region. The cardiac neural crest is located in a transitional area on the neuraxis between trunk and cephalic regions and gives rise to both the dorsolateral and ventrolateral crest cell populations. Around stage 18 of chick development, a mass of E/C8+ cells surrounds the postotic pharyngeal arches and forms a crescent-shaped arch, termed the circumpharyngeal ridge. Using immunohistochemistry and quail-chick chimeras, it was determined that the E/C8+ cell mass located in the circumpharyngeal ridge derives from the dorsolateral component of the cardiac neural crest. The ventrolateral cell population of the cardiac crest is located more medially and shows long-persistent HNK-1 immunoreactivity dorsolateral to the foregut. The crest cells that populate the gut arise from the caudal portion of the circumpharyngeal crest and are always located caudal to the caudal-most pharyngeal ectomesenchyme. Circumpharyngeal crest cells continuously populate the pharyngeal arch ectomesenchyme and enteric nervous system on the lateral side of the foregut wall, as well as the hypoglossal pathway which develops within the ventral portion of the circumpharyngeal ridge. E/C8 and HNK-1 immunoreactivity are associated with the cells migrating via the dorsolateral (circumpharyngeal) and ventrolateral pathways, respectively, with one exception: there is a population of putative crest cells along the proximal course of the vagal intestinal branch that shows both immunoreactivities around stage 20. DiI labeling of the cells in the circumpharyngeal ridge suggests that the cells are contributed from the circumpharyngeal ridge to this population. Thus, the distribution of the circumpharyngeal crest cells and their derivatives coincides with the peripheral branch distribution of the cranial nerves IX, X, and XII, whose development is selectively affected in the absence of the cardiac neural crest, the source of the circumpharyngeal crest.


Epidural patient-controlled analgesia: influence of bupivacaine and hydromorphone basal infusion on pain control after cesarean delivery. Epidural administration of hydromorphone was evaluated using a patient-controlled analgesia (PCA) delivery system in 170 healthy women undergoing elective cesarean delivery with epidural bupivacaine who were randomly assigned to one of four epidural PCA treatment groups: group I, hydromorphone alone by bolus administration; group II, hydromorphone, with a continuous (basal) infusion; group III, hydromorphone in combination with 0.08% bupivacaine by bolus administration; or group IV, hydromorphone and bupivacaine, with a concurrent infusion of both drugs. Patients in group I required significantly less opioid medication (2.1 +/- 1.1 mg [mean +/- SD]) during the first 24 h than patients in group II (3.3 +/- 1.3 mg). Similarly, patients in group III self-administered significantly less hydromorphone (2.0 +/- 1.0 mg) and bupivacaine (23.3 +/- 11.4 mg) during the first 24 h of PCA therapy, compared with patients in group IV (hydromorphone [2.7 +/- 1.1 mg] and bupivacaine [31.5 +/- 11.6 mg]). The concomitant use of a local anesthetic or basal opioid infusion with hydromorphone via epidural PCA did not decrease the number of PCA demands or delivered doses. In addition, patients in all four groups had similar pain, sedation, discomfort, fatigue, and anxiety scores. The frequency of awakening at night to self-administer analgesic medication was not decreased when a basal infusion was used.(ABSTRACT TRUNCATED AT 250 WORDS)


Free polymerized hemoglobin versus hydroxyethyl starch in resuscitation of hypovolemic dogs. Polymerized bovine hemoglobin (PBH) was compared with hydroxyethyl starch (HES) in a hypovolemic shock model. Eighteen dogs were subjected to hemorrhage; systolic arterial blood pressure was maintained at 40 mm Hg for 30 min (mean blood pressure 37.8 +/- 4.7 [SD] mm Hg). Resuscitation was conducted by infusing their own shed blood (control group) or 6% HES (mol wt 200,000) in 0.9% NaCl (HES group) or PBH (PBH group), both in an equal amount to the shed blood. Directly after infusion, oxygen delivery and consumption returned to prehemorrhage levels in all three groups. In the HES group, the lowered arterial oxygen content was compensated by a 158% increase in cardiac output, in contrast to an increase of 31% and 9%, respectively, in the control and PBH groups. Early recovery from hypovolemic shock with regard to oxygen transport and delivery in the PBH group seemed to be comparable to the control group, without the increase in cardiac output seen with HES infusion.


Nitric oxide synthase inhibitor dose-dependently and reversibly reduces the threshold for halothane anesthesia. A role for nitric oxide in mediating consciousness? Nitric oxide is a newly recognized cell messenger for the activation of soluble guanylate cyclase and is produced from L-arginine by the enzyme nitric oxide synthase in a wide variety of tissues, including vascular endothelium and brain. Inhalational anesthetics inhibit nitric oxide production from vascular endothelium and also decrease resting cyclic guanosine monophosphate content in multiple brain regions. Halothane has been shown to depress neurotransmission by L-glutamate and N-methyl-D-aspartate. These amino acid neurotransmitters are known to increase neuronal cyclic guanosine monophosphate content by stimulation of nitric oxide production. To investigate the possible involvement of the L-arginine-to-nitric oxide pathway in the anesthetic state, the effect of a specific nitric oxide synthase inhibitor, nitroG-L-arginine methyl ester, on the minimum alveolar concentration (MAC) for halothane anesthesia was determined in Sprague-Dawley rats. Bolus injection of nitroG-L-arginine methyl ester at 0, 1, 5, 10, 20, and 30 mg/kg resulted in a dose-dependent reduction in MAC for halothane of 0 +/- 0, 2.3 +/- 0.4, 21.5 +/- 3.9, 30.5 +/- 2.4, 51.0 +/- 7.8, and 26.0 +/- 2.8%, respectively. NitroG-L-arginine methyl ester had no effect on MAC for halothane. Bolus infusion of L-arginine 300 mg/kg after MAC reduction by nitroG-L-arginine methyl ester 10 mg/kg resulted in an immediate and complete reversal of the MAC reduction. No reversal was observed after infusion of D-arginine 300 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)


Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies. Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.


A model of ion channel kinetics based on deterministic, chaotic motion in a potential with two local minima. Models of the gating of ion channels have usually assumed that the switching between the open and closed states is a random process without a mechanistic basis. We explored the properties of a deterministic model of channel gating based on a chaotic dynamic system. The channel is modeled as a nonlinear oscillator, that has a potential function with two minima, which correspond to the stable open and closed states, and is driven by a periodic driving force. The properties of the model are like some properties of single channel data and unlike other properties. The model is like the data in that: the current switches between two well-defined states, this switching is nonperiodic, and there are subconductance states. These subconductance states are subharmonic resonances, due to the nonlinearities in the equation of the model, rather than stable conformational states due to local minima in the potential energy. The model is not like the data in that the current fluctuates too much within in each state and there are sometimes periodic fluctuations within a state. At the present time, the selection of the most appropriate channel model (Markov, chaotic, or other) is not possible, and in addition to chaotic models, other nonlinear models may be suitable.


Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5. Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.


Morphologic and cytochemical characteristics of blood cells from the desert tortoise (Gopherus agassizii). Morphologic and cytochemical staining characteristics of erythrocytes, leukocytes, and thrombocytes of the desert tortoise (Gopherus agassizii) were evaluated, using blood smears prepared from 23 healthy tortoises of Kern County, Calif. Special emphasis was placed on differentiating features of the various leukocytes and thrombocytes. A variety of cytochemical stains, including benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff, and toluidine blue were used. Heterophils had a characteristic, large, focal area of positive staining with chloroacetate esterase, alpha-naphthyl butyrate esterase, and acid phosphatase. Eosinophils stained diffusely positive with benzidine peroxidase, allowing differentiation of this leukocyte from heterophils. Thrombocytes stained focally positive with periodic acid-Schiff, allowing differentiation of these cells from lymphocytes, which stained uniformly negative. An intracytoplasmic body, commonly observed within erythrocytes, was considered ultrastructurally to represent a degenerate organelle.


[The role of lactate acidosis and enzyme hyperactivity in evaluating hypoxic organ disorders in patients following cardiopulmonary byøass]. One hundred and fifty patients after cardiopulmonary bypass surgery have been examined. It has been shown that overall enzyme activity should be taken into consideration when determining the genesis of postoperative hyperenzymemia and diagnosing the topical (organic) damage. High lactate level (6-8 mmol/l) reflects profound circulatory hypoxia and shows a great likelihood of secondary damage of parenchymatous organs in patients after cardiopulmonary bypass surgery. Marked lactatacidosis combined with a 5-10-fold increase in enzyme activity in the early postoperative period is an unfavourable prognostic sign. Prolonged antagosan (trasilol) administration during cardiopulmonary bypass surgery may be one of the techniques preventing and correcting hypoxic organ damage.


Mechanism of tetracycline-hydrochloride-induced pleurodesis. Tetracycline-hydrochloride-stimulated mesothelial cells produce a growth-factor-like activity for fibroblasts. Intrapleural instillation of tetracycline hydrochloride (TCN) is an effective means of achieving pleural fibrosis. However, its mechanism of action remains unknown. To evaluate the hypothesis that TCN stimulates pleural mesothelial cells to release growth-factor-like activity for fibroblasts we performed the following experiments. Rat visceral pleural mesothelial cells were incubated with TCN at doses ranging from 0.01 microgram/ml to 100 mg/ml. The conditioned media (CM) were collected after incubation for 2 to 48 h. CM caused fibroblasts to increase incorporation of thymidine when compared with CM that was unexposed to TCN (p less than 0.05). This growth-factor-like activity continued to be produced by mesothelial cells for 48 h after removal of TCN from the medium. There was a dose-response relationship since increasing doses of TCN to as much as 1 mg/ml caused increasing production of growth-factor-like activity without mesothelial cell injury as measured by trypan blue exclusion. The growth factor activity was a competence-type activity. It coeluted with human PDGF at a molecular weight of 31,000. It was heat-stable (100 degrees C for 10 min) and sensitive to trypsin and papain but not to heat-inactivated trypsin. Addition of cycloheximide or actinomycin D inhibited its production. TCN did not have any direct effect on fibroblasts. Bleomycin CM did not contain growth-factor-like activity for fibroblasts. These data demonstrate that TCN stimulates mesothelial cells to release a growth-factor-like activity for fibroblasts. This phenomenon may play an important role in TCN-induced pleural fibrosis.


Pharmacologic and neurochemical evidence for the activation of capsaicin-sensitive sensory nerves by lipoxin A4 in guinea pig bronchus. Exogenous administration of lipoxin A4 (LXA4) to guinea pig isolated bronchus produced contractile effects in a concentration-dependent manner (1, 3, and 6 microM). These responses were potentiated when preparations were previously incubated with thiorphan (10 microM), an inhibitor of tachykinin breakdown, but were significantly depressed when sensory nerves were previously desensitized in vitro by capsaicin (10 microM for 15 min) challenge. Ruthenium red (10 microM for 20 min), a blocker of the cationic channel coupled to the capsaicin receptor, also produced, although in a weaker manner, a reduction in bronchomotor responses elicited by LXA4. On the other hand, preexposure to omega-conotoxin (0.1 microM for 45 min), a blocker of neuronal voltage-dependent Ca2+ channels, did not modify the LXA4 contractile effects. Furthermore, LXA4 (6 microM) superfusion of guinea pig bronchial tissue elicited a significant calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release that was reduced by capsaicin (10 microM, 30 min) desensitization. Finally, LXA4 (10 microM) was unable to displace [3H]resiniferatoxin binding in dorsal root ganglion of rat and guinea pig. These findings support (1) a role for LXA4 in activating motor sensory function of capsaicin-sensitive nerves; (2) this activation mechanism is marginally ruthenium red-sensitive and omega-conotoxin-resistant; and (3) the interaction does not involve the recognized binding site on the vanilloid receptor. As a whole this study presents LXA4 as an endogenous mediator activating sensory nerves potentially involved in basic mechanisms of airway diseases.


Early markers of pancreas transplant rejection. To compare the predictive value of urinary amylase (UA), urinary insulin (UI), and urinary prostaglandin (PGE2), whole pancreas isografts or allografts from (ACI rat donors, RT1a) with bladder drainage of exocrine secretions were performed in Lewis rats (RT1(1)) with streptozotocin-induced diabetes. UA, UI, PGE2 and plasma glucose levels were measured daily. Euglycemia was restored on Postoperative Day 1 in all the recipients of isografts (N = 6) and was maintained for over a year. UI concentrations and PGE2 outputs were stable, with low levels ranging between 0.3 +/- 0.2 to 7 +/- 2 ng/ml and 56 +/- 15 to 164 +/- 48 ng/24 hr, respectively, while UA levels were significantly elevated compared to normal controls (> 1,000 U/ml vs 29 +/- 16 U/ml). In the allograft group (N = 12), rejection occurred on Days 7 through 9, with a mean graft survival time of 8.1 +/- 0.1 days. UA dropped from a post-transplant peak of 2,422 +/- 353 U/ml on Postoperative Day 4 to below 1,380 +/- 256 U/ml 3 days before rejection (Day -3). UI increased to 83 +/- 16 ng/ml (P < 0.05) on Day -6 and reached a post-transplant peak of 140 +/- 24 ng/ml on Day -5, while PGE2 output rose from a pretransplant level of 18 +/- 2 to 92 +/- 25 ng/24 hr on Postoperative Day 1, followed by a significant elevation on Day 4 (-4).(ABSTRACT TRUNCATED AT 250 WORDS)


[Clinical polymorphism of the XYY syndrome]. The XYY syndrome presents with a wide variation in the clinical features, both of the physical and behavioral nature. We report two new cases which illustrate this statement. The first case presented with aggressive behaviour and cryptorchidism. The second case was associated with pathological short height, pubertal delay and cardiac features (extrasystoles and short PR interval). We revise some of the aspects of XYY syndrome.


The premature ventricular complex as a diagnostic aid. Premature ventricular complexes (PVCs) can provide clues to the physical or electrocardiographic diagnosis through the associated compensatory pause, the break in the regularity of the rhythm, or the morphology of the PVC itself. A PVC may allow visualization of the P wave or of atrial flutter waves that would otherwise be obscured in the electrocardiogram. It can also be useful in distinguishing an S3 gallop from an S4 gallop. The compensatory pause that follows a PVC may allow normal conduction of the next QRS complex in a patient with a rate-dependent intraventricular conduction defect, and this normalized QRS complex may contain important diagnostic findings. A PVC can also reveal a myocardial infarct pattern when the sinus complex fails to do so. Although the need to treat PVCs is currently being de-emphasized, their diagnostic utility should not be overlooked.


Type I allergy in the inner ear of the guinea pig. Guinea pigs were passively sensitized by sera containing antidinitrophenyl reaginic antibody and specifically challenged by dinitrophenyl-bovine serum albumin injected through the stylomastoid foramen. Nystagmus, head deviation, negative summating potentials on electrocochleography, and an increase of threshold and wave I peak latency on auditory brain stem response testing were observed after local challenge. These physiologic changes were reversible and resolved within several days. We also used Tranilast before the specific challenge. It is a blocking agent of chemical mediator release from mast cells. Negative summating potentials and head deviation were not observed after the use of this agent. In the animals that showed physiologic changes, we observed endolymphatic hydrops, mast cell degranulation, and eosinophil infiltration histologically in the challenged side of the inner ear. These results suggest that the physiologic and histologic changes provoked in the inner ear of the sensitized animals may have been induced by type I allergy.


Plasma amylase estimation in recurrent abdominal pain in children. In a prospective study of 50 children who were admitted on more than one occasion with undiagnosed abdominal pain, the serum amylase was found to be normal in every case. One case of acute pancreatitis was diagnosed in over 8000 admissions. Serum amylase estimation did not contribute to the management of children with recurrent abdominal pain, and acute pancreatitis is so rare that routine amylase estimations cannot be recommended in paediatric surgical practice.


Should endoscopic stenting be the initial treatment of malignant biliary obstruction? Forty-two patients with biliary obstruction caused by a stricture had a diagnostic ERCP with subsequent insertion of a straight 10G endoprosthesis. These patients represented 70% of a cohort in which stent insertion had been attempted. The majority (63%) had pancreatic carcinoma, but 22% had malignant hilar obstruction. Five patients (12%) died within a few days of stent insertion; ERCP may have contributed to two deaths. Jaundice was relieved in all survivors. Median hospital stay was 6 days (range 2-32 days). After further investigation, nine patients were thought to be potentially curable and underwent laparotomy. Late complications after stent insertion alone included cholangitis (26%) and recurrent jaundice (28%). Only one patient developed gastric outlet obstruction and needed a gastroenterostomy. Median survival in the endoprosthesis group was 11 weeks (range 2-84 weeks). Survival was longer for patients with bile duct (14 weeks) rather than hilar strictures (6 weeks). Median survival after subsequent surgery was 40 weeks (range 4-80 weeks) with two long-term survivors. This study confirms that ERCP and stent insertion is a useful initial treatment for obstructive jaundice due to a biliary stricture, being both diagnostic and therapeutic. Subsequent evaluation for curative surgery is not precluded and in the majority of cases worthwhile palliation may be achieved by stenting alone.


A novel neuronal messenger molecule in brain: the free radical, nitric oxide. Understanding of the organization and function of a newly identified neuronal messenger molecule, nitric oxide, has progressed rapidly. Nitric oxide synthase has been purified and molecularly cloned from brain. Its localization is exclusively neuronal and endothelial. The catalytic activity of nitric oxide synthase accounts for the NADPH diaphorase staining of neurons that are uniquely resistant to toxic insults and neurodegenerative disorders. Nitric oxide has diverse functions. In platelets it inhibits their aggregation, in macrophages it mediates cytotoxicity, and in blood vessels it acts as a vasodilator. In the nervous system nitric oxide may be the retrograde transmitter in long-term potentiation. It is the "neurotransmitter" of cerebral vasodilator nerves and the inhibitory "neurotransmitter" of the motor neurons of the intestines. Nitric oxide in situations of excessive production may function as a neurotoxin, suggesting a role for nitric oxide in neurodegenerative disorders.


Myelin basic protein-specific T lymphocytes in multiple sclerosis and controls: precursor frequency, fine specificity, and cytotoxicity. A panel of 90 myelin basic protein (MBP)-specific T-cell lines were derived from peripheral blood of eight patients with multiple sclerosis and four normal subjects. The precursor frequency of MBP-reactive T cells in peripheral blood mononuclear cells ranged from 10(-7) to 9 x 10(-7) (mean, 6.7 x 10(-7)) in the group of patients with multiple sclerosis and from 0.5 x 10(-7) to 9.8 x 10(-7) (mean, 5.6 x 10(-7)) in the control subjects. This difference between the two groups was not statistically significant (p greater than 0.1). These T-cell lines expressed exclusively CD3+CD4+CD8- phenotypes and were restricted predominantly by HLA-DR molecules. When tested with fragments and synthetic peptides of human MBP, these MBP-specific T-cell lines (45 lines for each group) displayed a limited heterogeneous pattern with a biased recognition to peptide 84-102 and the C-terminal peptide 149-171. The reactivity to the 84-102 region of MBP was associated with the HLA-DR2, DRw15 (DRw15,2) haplotype, whereas the recognition to peptide 149-171 did not correlate with a particular HLA-DR allele(s). Furthermore, the majority of T-cell lines (greater than 75%) were found to exhibit substantial cytotoxic activity against MBP-coated target cells, but showing no significant difference between these two groups. This MBP-dependent cytotoxicity was not associated with epitope specificities of the T-cell lines tested.


Bacterial toxin superantigens activate human T lymphocytes reactive with myelin autoantigens. Some bacteria that are common human pathogens produce protein toxins that are potent activators of human T lymphocytes expressing certain types of T-cell receptors. In this study we examined the ability of staphylococcal toxins to stimulate human T lymphocytes that also recognized the myelin autoantigens myelin basic protein and proteolipid protein. T-cell populations responding to myelin basic protein or proteolipid protein were isolated from 4 subjects including 1 individual with multiple sclerosis. All myelin antigen-specific T cells responded in proliferation studies to at least one of the nine superantigenic toxins used in this study. The superantigenic toxins were up to 7 x 10(5)-fold more potent in proliferation assays than the myelin antigens to which the T cells were initially sensitized. In addition, cytotoxic, myelin basic protein-reactive T lymphocytes lysed antigen-presenting cells incubated with superantigenic toxins. These findings demonstrate a mechanism by which some bacterial infections might produce activation of myelin basic protein- and proteolipid protein-reactive T lymphocytes and perhaps contribute to demyelinating disease in humans.


Absence of "red man syndrome" in patients being treated with vancomycin or high-dose teicoplanin. Twenty-five febrile patients with a history of intravenous drug use who were receiving either vancomycin (15 patients) or teicoplanin (10 patients) as part of a multicenter, double-blind, randomized, clinical efficacy trial were enrolled, upon receipt of their first dose of antibiotic, into a study to evaluate the effect of 1 g of vancomycin and high-dose teicoplanin (30 mg/kg of body weight) on histamine release and the occurrence of "red man syndrome" (RMS). In addition, 10 healthy volunteer subjects (HVS) were randomized to receive either 1 g of vancomycin intravenously or a saline infusion in a double-blind, crossover design study. Patients and HVS were observed for the presence of erythema, flushing, pruritus, and hypotension during and for up to 1 h postinfusion by a blinded investigator. Histamine concentrations in plasma were measured at baseline and during and after drug infusion. No significant differences were noted in baseline temperature between patients (vancomycin recipients, 102.3 degrees F [39.1 degrees C]; teicoplanin recipients, 102.4 degrees F [39.1 degrees C]) or incidence of bacteremia (7 of 15 vancomycin recipients; 5 of 10 teicoplanin recipients). There were no significant differences in peak vancomycin concentrations in the sera of patients (40.8 micrograms/ml) and HVS (49.9 micrograms/ml). There were no reactions consistent with RMS in any patient who received teicoplanin (0 of 10); there was a significant difference in the occurrence of RMS in patients in comparison with that in HVS (0 of 15 patients, 9 of 10 HVS; P less than 0.001) who received vancomycin. The predominant reaction was erythema and pruritus. Histamine concentrations in plasma and the area under the histamine plasma concentration-time curve were highly variable within groups and were not statistically different between patients and HVS. The incidence of RMS secondary to vancomycin or teicoplanin in our patient population appears to be low and consistent with clinical observations. Similar to previous investigations, RMS secondary to vancomycin in HVS was high (90%). However, we found no relationship between the histamine concentration in plasma or the area under the plasma histamine concentration-time curve and the severity of RMS in HVS. The reason for the discrepancy of RMS in patients versus that in HVS in unknown, but it may be related to a blunted effect of glycopeptides to produce the reaction in the presence of infection or it may be specific to our patient population.


Anti-human immunodeficiency virus activity of a novel synthetic peptide, T22 ([Tyr-5,12, Lys-7]polyphemusin II): a possible inhibitor of virus-cell fusion. More than 40 peptides associated with tachyplesin and polyphemusin, which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus, were synthesized. Among these peptides, we found that a novel compound, which was called T22 ([Tyr-5,12, Lys-7]polyphemusin II), strongly inhibited the human immunodeficiency virus type 1 (HIV-1)-induced cytopathic effect and viral antigen expression. Its 50% effective concentration was 0.008 micrograms/ml, while its 50% cytotoxic concentration was 54 micrograms/ml. The anti-HIV activity of T22 was observed with several strains of HIV-1, including zidovudine-resistant strains, and with HIV-2 within the concentration range of 0.006 to 0.071 microgram/ml. T22 efficiently inhibited giant cell formation on the cocultivation of MOLT-4/HIV and MOLT-4 cells but modestly inhibited direct HIV binding. T22 did not inhibit reverse transcriptase activity. A time-of-addition study, which involved monitoring of the appearance of proviral DNA by using the polymerase chain reaction technique, found that T22 exerted its effect on a process, most probably virus-cell fusion or uncoating, immediately after virus adsorption.


Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerases alpha, beta, and gamma. (-)-2'-Deoxy-3'-thiacytidine (3TC) is a selective inhibitor of human immunodeficiency virus replication in vitro (J. A. V. Coates, N. Cammack, H. J. Jenkinson, A. J. Jowett, M. I. Jowett, B. A. Pearson, C. R. Penn, P. L. Rouse, K. C. Viner, and J. M. Cameron, Antimicrob. Agents Chemother. 36:733-739, 1992). The effect of 3TC 5'-triphosphate on both the RNA-dependent and DNA-dependent activities of human immunodeficiency virus type 1 reverse transcriptase and DNA polymerases alpha, beta, and gamma from HeLa cells was investigated. 3TC 5'-triphosphate is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity (apparent Ki = 10.6 +/- 1.0 to 1.24 +/- 5.1 microM, depending on the template and primer used); the DNA-dependent DNA polymerase activity is 50% inhibited by a 3TC 5'-triphosphate concentration of 23.4 +/- 2.5 microM when dCTP is present at a concentration equal to its Km value. Chain elongation studies show that 3TC 5'-triphosphate is incorporated into newly synthesized DNA and that transcription is terminated in a manner identical to that found for ddCTP. The 50% inhibitory concentrations of 3TC 5'-triphosphate against DNA polymerases alpha, beta, and gamma at concentrations of dCTP equal to the Km were 175 +/- 31, 24.8 +/- 10.9, and 43.8 +/- 16.4 microM, respectively. More detailed kinetic studies with 3TC 5'-triphosphate and DNA polymerases beta and gamma are consistent with the fact that inhibition of these enzymes by 3TC 5'-triphosphate is competitive with respect to dCTP. The values of Ki were determined to be 18.7 microM for DNA polymerase beta and 15.8 +/- 0.8 microM for DNA polymerase gamma.


Methylation of HSV-1 DNA as a mechanism of viral inhibition: studies of an analogue of methyldeoxycytidine: trifluoromethyldeoxycytidine (F3mdCyd). Although several hypomethylating agents such as 5-azadeoxycytidine and 5-fluorodeoxycytidine have been shown to activate transcription after incorporation into viral or cellular DNA, agents which selectively affect the methylation status of virus-infected cells have not been described. Studies on the antiviral effect of the methyldeoxycytidine (mdCyd) analogue trifluoromethyldeoxycytidine (F3mdCyd) showed significant antiviral activity against herpes simplex virus type 1 (HSV-1). This analogue of both dCyd and dThd is selectively incorporated into the DNA of herpesvirus infected cells due to the unique specificity of the herpesvirus thymidine kinase (TK) because the HSV-1 TK is both a dCyd and dThd kinase. In contrast, the deoxycytidine kinase of uninfected cells preferentially phosphorylates dCyd and has a poor affinity for F3mdCyd. F3mdCyd hemisubstituted M13 DNA displayed the same properties as mdCyd-substituted M13 DNA with respect to cleavage by restriction enzymes, and acted as an efficient template for eukaryotic DNA methyltransferase (S-adenosyl-L-methionine DNA (cytosine-5) methyltransferase: EC 2.1.1.37). Using the persistently infected CEM cell model system, the extent of DNA methylation was shown to increase in a dose-related manner when HSV-1-infected CEM cells were treated with increasing concentrations of F3mdCyd. Higher levels of methylation correlated with significant decreases in HSV-1 titers. Isoschizomer analyses followed by Southern blotting and hybridization with genomic HSV-1 DNA showed that DNA from HSV-1-infected, analogue-treated Vero cells was resistant to cleavage by restriction enzymes at a time when productive virus was not present in culture. We infer from these results that the methylation-like properties of the incorporated F3mdCyd occur concomitantly with, and appear to be involved in, the mechanisms of the analogue's antiviral effect towards HSV-1.


In vitro selection of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine. 3'-Azido-3'-deoxythymidine (AZT)-resistant human immunodeficiency virus type 1 (HIV-1) was obtained by growing HTLV-IIIB in C8166 cell cultures in the presence of inhibitory concentrations of AZT. The AZT-resistant HIV-1 was capable of replicating, as measured by infectious virus yield, and inducing cytopathic effect in the presence of AZT concentrations able to completely suppress the replication of parental HTLV-IIIB. Cloning of the AZT-resistant HIV-1 revealed that a number of different variants of HIV-1 with various degrees of sensitivity to AZT emerged during propagation of HTLV-IIIB in C8166 cells in the presence of the drug. PCR experiments performed on DNA extracted from C8166 cells infected with a resistant strain revealed that viral DNA was produced in the presence of inhibitory concentrations of AZT, while viral DNA in C8166 cells infected with the parental virus was drastically inhibited. Reverse transcriptase isolated from the AZT-resistant HIV-1 variant failed to show resistance to AZT 5'-triphosphate.


Sulfonic acid polymers as a new class of human immunodeficiency virus inhibitors. Four sulfonic acid polymers [poly(4-styrenesulfonic acid)(PSS), poly(anetholesulfonic acid)(PAS), poly(vinylsulfonic acid)(PVS), poly(2-acrylamido-2-methyl-1-propanesulfonic acid)(PAMPS)] have been found to inhibit the cytopathicity of HIV-1 and HIV-2 in MT-4 cells at concentrations that are not toxic to the host cells. The sulfonic acid polymers also inhibited syncytium formation in co-cultures of MOLT-4 cells with HIV-1- or HIV-2-infected HUT-78 cells. They also inhibited binding of anti-gp120 mAb to HIV-1 gp120 and blocked adsorption of HIV-1 virions to MT-4 cells. PSS and PAS, but not PVS and PAMPS, interfered with the binding of OKT4A/Leu3a to the CD4 receptor. The anti-HIV activity of these polyanionic compounds can be ascribed to inhibition of the gp120-CD4 interaction. Sulfonic acid polymers represent a lead of anti-HIV compounds that warrant further evaluation of their therapeutic potential.


Post-hospitalization concerns of medical-surgical patients. In response to the phenomenon of earlier-than-ever discharge, the post-hospitalization concerns of medical-surgical patients were investigated in a descriptive survey conducted in physicians' offices and clinics. The 150 recently discharged patients typically identified multiple concerns including understanding their progress, activity, insurance, medications, and pain control. Differences were found in number of concerns according to age, gender, length of hospital stay, type of treatment (medical or surgical), and perceived presence or absence of discharge planning.


Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma. The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.


Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells. Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.


Dual effects of oleic acid on Ca2+ mobilization and protein phosphorylation in human platelets in presence or absence of platelet activating factor. This laboratory demonstrated earlier that oleic acid inhibited platelet activating factor (PAF)-induced aggregation and serotonin release of rabbit platelets (M. Miwa, C. Hill, R. Kumar, J. Sugatani, M. S. Olson, and D. J. Hanahan, 1987, J. Biol. Chem. 262, 527-530). More recently, we reported that oleic acid caused a decrease in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), but did not affect the level of inositol-1,4,5-trisphosphate (IP3), in rabbit platelets (D. Nunez, J. Randon, C. Gandhi, A. Siafaka-Kapadai, M. S. Olson, and D. J. Hanahan, 1990, J. Biol. Chem. 265, 18330-18838). These results suggested that oleic acid did not stimulate phospholipase C. In contrast, PAF induced a decrease in PIP2 and an increase in PIP level and IP3. These effects were shown to be attenuated by oleic acid. In this current study, our experiments show that (a) oleic acid blocked PAF-induced rise in intracellular [Ca2+] (to provide a mechanism in agreement with our previous experiments which showed that oleic acid inhibited PAF-induced IP3 rise in platelets) and (b) oleic acid itself induced a gradual rise in [Ca2+]i, which would provide a mechanism for oleic acid-induced aggregation despite the fact that oleic acid did not cause the production of IP3 (Nunez et al., 1990). Oleic acid, in a dose-dependent manner, was shown to inhibit PAF-induced Ca2+ mobilization from intra- and extracellular sources. The inhibition was closely related to the suppressive effect of oleic acid on PAF-induced aggregation. Furthermore, oleic acid inhibited the PAF-stimulated phosphorylation of the 20- and 40-kDa proteins. At concentrations above 20 microM, oleic acid itself could induce platelet aggregation and Ca2+ mobilization, but the time sequence of these two responses in human platelets was significantly different from those obtained with PAF. Oleic acid alone, at 20 microM, caused a 1.4-fold increase in the cAMP level in platelets which was followed by a decline to a basal value at higher concentrations of this fatty acid. It seemed clear that elevation of adenylate cyclase activity was not associated with free fatty acid inhibition of platelet activation. Interestingly, both PAF and oleic acid added separately to human platelets induced protein-tyrosine phosphorylation, but oleic acid did not cause any inhibition of PAF-induced protein-tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)


Rat cystathionine beta-synthase: expression of four alternatively spliced isoforms in transfected cultured cells. The gene for rat cystathionine beta-synthase consists of 17 exons. Its transcripts are alternatively spliced, forming four distinct mRNA species. Type III consists of exons 1 through 12, 14, 15, and 17; type I also contains exon 16. The open reading frame of type IV spans exons 1-->13; type II, 3-->13. We cloned the corresponding cDNAs into appropriate expression vectors and inserted the constructs into Escherichia coli (I, III, and IV) and Chinese hamster (CH) cells (I through IV); all sequences were transcribed and translated. Catalytic activity was observed only for types I and III in lysates of transfected CH cells and transformed E. coli. The catalytic and kinetic properties of I and III were identical despite their structural difference (exon 16). Both isoforms exhibited 6 mM Km constants for homocysteine which were reduced approximately eightfold by AdoMet; this elucidates the mechanism by which AdoMet regulates synthase activity. The four isoforms were differentially degraded by transfected cultured cells. Type III (t1/2 = 18 h) was degraded at 1/3 the rate of type I (t1/2 = 6 h); thus the 14 amino acid residues encoded by exon 16 appear to enhance degradation of CBS. The half-lives of both types II and IV were markedly shorter (ca. 1 h). Western blots comparing rat liver to lysates from transfected CH cells revealed that hepatocytes express both isoforms. Type III was predominant, as predicted by its longer half-life and more abundant mRNA. PCR analysis of cDNA from various tissues revealed that type III mRNA was preferred in liver, kidney, and heart; equal amounts of I and III were found in brain.


Hexachlorocyclohexane pesticides reduce survival and alter plasma membrane structure of Chinese hamster V79 cells. We studied the cytotoxic effects of alpha-, beta-, gamma-, and delta-hexachlorocyclohexanes (HCCH) on the survival of Chinese hamster V79 cells using clonogenic assays. Lethal dose yielding 50% cell survival (LD50) suggests the following order of cytotoxicity: delta-(+)gamma-HCCH (LD50 4 micrograms/ml) (1:1, w/w, mixture) > delta-HCCH (LD50 6 micrograms/ml) > gamma-HCCH (LD50 13 micrograms/ml) > alpha-HCCH (LD50 approx. 35 micrograms/ml) >> beta-HCCH. Structural changes in plasma membranes prepared from HCCH-treated V79 cells at dose yielding 10% cell survival (LD10) were analyzed using Raman spectroscopy. Raman spectra of plasma membranes show bands at 2850, 2880-2890, and 2935 cm-1 in the C-H stretching region. The plot of the ratio (I2880-2890/I2850) vs temperature for control plasma membranes shows two transitions between -5 and 5 degrees C and between 12 and 20 degrees C. Plasma membranes prepared from gamma- and delta-HCCH-treated Chinese hamster V79 cells show single transitions between -4 and 11 degrees C and between -2 and 11 degrees C, respectively. These changes in the thermal transition properties suggest that both gamma- and delta-HCCH alter lipid and lipid-protein phases of the plasma membrane of V79 cells. Raman analysis of the amide I and amide III region spectra further suggest that delta-HCCH also alters the secondary structure and the environment of highly amidated segments of plasma membrane proteins. We suggest that the primary action of biologically active HCCH isomers is to disrupt the organization of the plasma membrane and that may affect cell viability.


Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor. The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.


[Recent advances in ovarian cancer chemotherapy]. The standard approach for epithelial ovarian cancer has been maximum cytoreductive surgery followed by combination therapy. Several prospective control studies individually failed to demonstrate improved survival advantage for the Adriamycin containing combination compare with cisplatin plus cyclophosphamide. The two drug combination of carboplatin plus cyclophosphamide will be thought to become the treatment of choice, because it is equally effective as and less toxic than a regimen of cisplatin plus cyclophosphamide. Clinical trials are also in progress with more dose-intense regimens based on considerable retrospective evidence that survival is correlated with the dose intensity of platinum compounds. Currently, high dose carboplatin plus Gm-CSF, two-drug combination of carboplatin and cisplatin and super high dose carboplatin combined with autologous bone marrow transplantation are undergoing clinical trials. Taxol and taxotere, most important cancer drugs after emergence of cisplatin compound, has been shown to have clinical activity in drug resistant ovarian cancer patients. Majority of patients even with advanced germ cell tumors of the ovary is now cured because of the development of effective platinum-based combination chemotherapy of PVB or BEP.


[Symptomatic treatment of benign hypertrophy of the prostate. Comparative study of prazosin and serenoa repens]. Forty-five patients diagnosed as having BPH and clinically diagnosed micturition disorders were entered in a therapeutic protocol. Twenty-five patients received Prazosin and the remaining 20 patients were treated with Serenoa Repens for a period of 12 weeks. The symptomatology was assessed by flowmetry and the patients were questioned as to the irritative symptoms. It can be concluded from the study that Prazosin is slightly more effective in controlling the irritative symptoms produced by BPH.


Efficacy of eight serially measured markers for diagnosis of prostatic carcinoma. In this study we measured eight different tumor markers (PSA, PAP, TPA, CEA, Ca 50, Ca 19-9, Ca 125 and Ca 15-3) in 39 patients with prostatic adenocarcinoma and in 90 patients with benign prostatic hyperplasia. We then calculated the sensitivity and specificity for each tumor marker separately and found that only PSA, when we consider as normal value 10 ng/ml, has a sufficiently high sensitivity and specificity. Our conclusion is that only PSA can be used for diagnostic purposes in conjunction with other diagnostic modalities.


Characteristics of immunocompetent cells in synovial membranes from multiple sites in patients with rheumatoid arthritis. The phenotypic characterization of enzymatically dissociated mononuclear cells in synovial membrane samples from multiple sites in two patients with rheumatoid arthritis (RA) were examined by fluorescence activated flow cytometry. In synovial membrane samples from each patient there was a consistent increase in the proportion of CD8+ cells (suppressor/cytotoxic), CD14+ cells (monocytes/macrophages), and HLA-DR+ cells compared with paired peripheral blood mononuclear cells. The proportion of CD4+ cells (helper/inducer) in synovial membrane was variable. Studies of in vitro production of IgM and IgM rheumatoid factor in one patient showed strikingly similar values for synovial membrane rheumatoid factor production at the two sites, which was enhanced compared with production in peripheral blood. These results suggest that in individual patients with RA the intra-articular immune response is comparable at multiple anatomical sites and that it is distinct from that in peripheral blood.


Intestinal transplantation in composite visceral grafts or alone. Under FK 506-based immunosuppression, the entire cadaver small bowel except for a few proximal and distal centimeters was translated to 17 randomly matched patients, of whom two had antigraft cytotoxic antibodies (positive cross-match). Eight patients received the intestine only, eight had intestine in continuity with the liver, and one received a full multivisceral graft that included the liver, stomach, and pancreas. One liver-intestine recipient died after an intestinal anastomotic leak, sepsis, and graft-versus-host disease. The other 16 patients are alive after 1 to 23 months, in one case after chronic rejection, graft removal, and retransplantation. Twelve of the patients have been liberated from total parenteral nutrition, including all whose transplantation was 2 months or longer ago. The grafts have supported good nutrition, and in children, have allowed growth and weight gain. Management of these patients has been difficult and often complicated, but the end result has been satisfactory in most cases, justifying further clinical trials. The convalescence of the eight patients receiving intestine only has been faster and more trouble free than after liver-intestine or multivisceral transplantation, with no greater difficulty in the control of rejection.


Exocrine pancreatic function in mediastinal teratomata: an aid to preoperative diagnosis? The diagnosis of teratoma may be made by demonstration of high amylase content in fluid aspirated from anterior mediastinal lesions. In 2 cases of mediastinal teratoma proteolytic enzyme activity was evident at the time of operation. A diagnosis of mediastinal teratoma was aided in 2 subsequent cases by demonstration of elevated amylase activity in the aspirated fluid before definitive operation.


Expanding wire stents in benign tracheobronchial disease: indications and complications. Prosthetic tracheobronchial stents provide palliative treatment for narrowed airways where surgical resection is inadvisable. Over a 1-year period, 28 Gianturco expanding wire stents were used in 15 patients for nonneoplastic indications: pure fibrous airway stenosis (6), fibroinflammatory stenosis (4), and tracheobronchial malacia (5). Insertion was technically straightforward. A satisfactory airway lumen with immediate improvement in ventilatory function was obtained in all patients. After insertion all patients had an irritation-type cough that either subsided spontaneously (10 patients) or was successfully suppressed with inhaled corticosteroid therapy (5 patients). The most common complication (12 patients) was granuloma formation leading to stent removal in 3 patients with fibroinflammatory stenosis. Other complications were dysphagia (1), suction catheter entrapment (1), and fatal massive hemoptysis (1). At a mean follow-up of 13 months (range, 3 to 19 months) all remaining stents are functioning well with no displacement or infection. Overall results were satisfactory in pure fibrous stenoses and tracheobronchial malacia but poor in the presence of inflammation. Tracheobronchial wire stents can be successfully used in selected patients.


Surgically created Wolff-Parkinson-White syndrome after Fontan operation. The Wolff-Parkinson-White syndrome is caused by a congenital accessory connection between the atrium and ventricle. We describe a case of symptomatic Wolff-Parkinson-White syndrome that arose after a Björk modification of the Fontan operation. Invasive electrophysiologic and intraoperative mapping indicated that the surgically created atrioventricular connection was functioning as an accessory pathway. Surgical dissection and cryoablation abolished the symptoms and the preexcitation.


[Study of possible biological transformation of kanamycin to amikacin]. For transformation of kanamycin A (Km) to amikacin (Ak) with acylating enzymes from B. circulans, a culture producing butirosin (Btn), cellular and acellular conversion systems and methods for chemical and biological identification of Km, Ak and Btn were developed. The level of conversion of Km to Ak in vivo and in vitro did not exceed 2 per cent.


[Selection and physiological study of culture of Bacillus circulans-- producer of butirosin]. For isolating a highly active variant of the butirosin-producing culture, a strain forming trace amounts of the antibiotic substance was used. Exposure to nitrosomethylbiuret and nitrosoguanidine and the use of selective media containing streptomycin and butirosin resulted in a 30-fold increase in the strain productivity. Thin layer chromatography of the produced antibiotic substance in the solvent system developed by the authors, mass spectrometry and assay of the antimicrobial spectrum in regard to ++gram-positive and ++gram-negative bacteria by using the known aminoglycosidine-inactivating enzymes revealed that the substance was identical to butirosin. Along with the major product, the fermentation broth contained up to 5 per cent of ribostamycin.


Cystadenoma and cystadenocarcinoma with mesenchymal stroma of the liver. Immunohistochemical analysis. Cystadenoma with mesenchymal stroma is a rare neoplasm of the liver that occurs exclusively in young women and has a potential for malignant transformation. A light microscopic and immunohistochemical study of a case of biliary cystadenoma and another of biliary cystadenocarcinoma revealed a range of differentiation of the lining epithelial cells. The lining cells in the cystadenoma resembled the cells of the normal intrahepatic bile ducts. In contrast, the epithelial lining in the case of cystadenocarcinoma had features of intestinal mucosa, including goblet, Paneth, and endocrine cells similar to those found in other mucinous cystic neoplasms of the foregut area. The compact "ovarianlike" mesenchymal stromal cells had immunohistochemical characteristics of myofibroblasts. These are reactive contractile cells that may proliferate in response to the expanding cysts and female hormones, and they differ immunohistochemically from ovarian stromal cells.


A modified Da Fano silver stain for demonstration of neurons and dendrites in glycol methacrylate-embedded brain tissue. Golgi impregnation techniques are commonly used for characterization of neurons and their dendritic and axonal processes. Most of the widely used techniques require processing of fresh brain tissues, which limits the amount of material available for study. Additionally, the stained blocks must be subsequently embedded in paraffin, which produces considerable cellular shrinkage and distortion artifacts. Modification by one investigator of an early silver impregnation technique, designed to demonstrate the Golgi apparatus, allowed demonstration of neurons and their dendritic processes. Our further modification of the later technique, along with embedding of the stained tissue in glycol methacrylate, permits detailed examination of neurons and their processes in formaldehyde-fixed neonatal human brains. In cerebellar sections, this modified technique impregnates nearly all Purkinje cells, elucidating the fine structural detail of the developing neuronal dendritic tree and spines.


Expression of P-glycoprotein in normal muscle cells and myogenic tumors. We have analyzed the expression of the multidrug resistance (mdr-1) gene product, P-glycoprotein, by immunohistochemical staining of frozen tissue sections of human normal muscle fibers and 31 tissue specimens of cases of myogenic sarcomas. The objective of this study was to further characterize what appears to be a variety of responses to therapy in like-appearing but distinct tumors. We have used two mouse monoclonal antibodies that recognize two different epitopes of P-glycoprotein. Mouse monoclonal antibody HYB-241 detects an extracellular epitope of P-glycoprotein, whereas C219 detects a carboxy-terminal intracellular epitope and has recently been reported to cross-react with the mdr-3 gene product. Differential epitope expression was observed among normal muscle fibers with the two antibodies used. Smooth-muscle cells were unreactive for the two antibodies, whereas cardiac and a subgroup of skeletal muscle fibers were intensely stained by C219, but not by HYB-241. P-glycoprotein expression was observed in 23% of the 31 myogenic sarcomas analyzed. Our study was conducted mainly using adult myogenic sarcomas (28 out of 31 cases), with a few cases (three out of 31 cases) of childhood sarcomas. Nineteen tumors were leiomyosarcomas, seven cases were embryonal rhabdomyosarcomas, and five cases were rhabdomyosarcomas. We have considered expression of the mdr-1-coded P-glycoprotein when we observed either HYB-241 and C219 staining, or just HYB-241 immunoreactivities. Although P-glycoprotein expression can now be detected in human sarcomas, further studies are needed, mainly comparing tumor samples before, during, and after therapy, to establish the possible significance of the P-glycoprotein expression in clinical drug resistance.


Hemangioendothelioma of the spleen.

Hemangioendothelioma of the spleen. Hemangioendotheliomas of the spleen are rare and are considered to be of intermediate/borderline malignancy. We report such a case in a patient who presented with chronic anemia but who otherwise was asymptomatic. The tumor involved half the organ and was solitary and nonencapsulated. Microscopically, it was composed of vascular and stromal elements. Both types of elements showed moderate atypia and rare mitoses. The lining cells stained positively with antibodies to factor VIII-related antigen and Ulex europaeus lectin. The stromal component showed evidence of myofibroblastic differentiation. One year after splenectomy, all hematologic parameters slowly improved and returned to normal. The clinicopathologic differences between hemangioma, angiosarcoma, and hemangioendothelioma are discussed, and cases that have recently been reported in the literature are reviewed.


Aorticopulmonary paraganglioma. A case report with immunohistochemical studies and literature review. We present a case of nonfunctioning aorticopulmonary paraganglioma in a 41-year-old man. The mediastinal tumor was accidentally discovered on a chest roentgenogram performed for an unrelated minor chest trauma. Complete resection of the tumor was done and followed by postoperative radiotherapy. Two years after surgery, the patient is well and asymptomatic.


Intracellular pH changes induced by exposure to weak acids and bases in submandibular cells of early postnatal rats. The intracellular pH of submandibular cells of adult, 1-day-old and 1-week-old rats was measured with the pH sensitive fluorimetric indicator SNARF-1, following shifts induced by exposure to a weak acid (sodium butyrate) or a weak base (NH4Cl). The effects of several ion transport inhibitors and agonists on both the pH change and the rate of recovery were examined. The results indicate that, in most respects, the processes involved in pH regulation were similar in the neonatal and mature cells. However, cholinergic and peptidergic stimulation accelerated recovery from sodium butyrate-induced acidification more, and from NH4Cl-induced alkalinization less, in cell preparations from 1-day-old animals.


Methionine enkephalin-like, substance P-like, and beta-endorphin-like immunoreactivity in human parotid saliva. These three neuropeptides were measured at daily baseline values by radioimmunoassay. Stimulated parotid saliva was collected from 31 subjects using a modified Carlson-Crittenden device affixed over Stenson's duct. Methionine enkephalin-like immunoreactivity ranged from 6.6 to 11.7 fmol/ml, with a mean of 9.3 fmol/ml. Substance P-like immunoreactivity ranged from 6.1 to 12.6 fmol/ml, with a mean of 9.3 fmol/ml. beta-Endorphin-like immunoreactivity ranged from 1.2 to 3.6 fmol/ml, with a mean of 2.6 fmol/ml. This is believed to be the first documentation of methionine enkephalin- and substance P-like activities in human parotid saliva and the first demonstration of beta-endorphin-like activity in any type of human saliva. Substance P-like activity was significantly higher in morning than evening samples; beta-endorphin-like activity also tended to be higher in the morning samples. Substance P and beta-endorphin-like immunoreactivities covaried in a significant positive manner, suggesting either common control mechanisms or similar responses to physiological variables.


Calcium channel modulators and susceptibility to ischaemic ventricular fibrillation: modification of cellular calcium overload. The effects of the calcium channel modulators, Bay k 8644, infused i.v. at a rate of 2.5 micrograms/kg/min, and diltiazem, injected i.v. in a dose of 0.5 mg/kg, on the susceptibility to fibrillation induced by ischaemia, were investigated in anaesthetized, open-chest pigs. Ischaemia was produced, under ventricular pacing at constant high rate (180 beats/min), by transient complete occlusion of the left anterior descending coronary artery, near its origin. It was maintained till the triggering of fibrillation. The propensity to fibrillation was judged from the time elapsing between the onset of occlusion and the onset of fibrillation (time to fibrillation). In addition to the surface electrocardiogram, conduction time and monophasic action potential were recorded in the ventricular contractile fibres, as were dP/dtmax in the left ventricle and blood pressure in the carotid artery. At the end of a 10 min infusion, Bay k 8644 lowered to a large extent (about 40%) the time to fibrillation, which returned to its control values within the following 20 min. Conversely, diltiazem increased the time to fibrillation by a factor 4 or 5 at 5 min after its administration. This time to fibrillation remained substantially increased 25 min later. These changes were not associated with alterations in conduction time or monophasic action potential duration in the absence of ischaemia, but with significant alterations in myocardial contractility and blood pressure: in the direction of an increase with Bay k 8644 and of a decrease with diltiazem. These results are in agreement with the enhancement by Bay k 8644 and the prevention by diltiazem of cell calcium overload which is at present recognized as being the essential determinant of the fibrillatory process.


Microrips of the retinal pigment epithelium. We describe six patients with exudative age-related macular degeneration who had retinal pigment epithelial detachments with associated overlying neurosensory detachments. During fluorescein angiography, each patient demonstrated a solitary, intense, central serouslike leak at the edge of the retinal pigment epithelial detachment with passage of fluorescein into the subretinal space. In patients in whom the location of associated choroidal neovascularization was evident, the leakage site was remote to the area of neovascularization. Our observations suggest that these leaks result from small retinal epithelial rips, which we termed "microrips," that differ from conventionally described retinal pigment epithelial rips in clinical course and response to laser treatment. We hypothesize that the mechanisms and forces that generate these microrips are different from those producing conventionally described retinal pigment epithelial rips.


Parathyroid hormone fragment 1-34 and anti-inflammatory effect. We have studied the possible activity of the rat parathyroid hormone fragment 1-34, peripherally or centrally injected, on the vascular permeability induced by local administration of croton oil. Our study considered also the possible interference of parathyroid hormone fragment 1-34 on the release of histamine by mast cells and on the content of histamine in paw tissue. Parathyroid hormone fragment 1-34 significantly reduced the increase in vascular permeability produced in mice by local croton oil injection, as well as the serotonin-induced paw edema in the rat. These effects of parathyroid hormone fragment 1-34 were present only after peripheral administration. At the dose of 3.3 micrograms/kg, it decreased the histamine content in paw tissue. Peripheral administration of a calcium antagonist, such as nimodipine, at the dose of 50 micrograms/kg, or a metal-chelating agent, such as ethylene-diaminetetraacetic acid, at the dose of 10 micrograms/kg, antagonized the anti-inflammatory effect of parathyroid hormone fragment 1-34. Moreover, at 10-100 microliters/ml it significantly decreased the histamine release by mast cells.


Fusion between rat pancreatic zymogen granules and plasma membranes. Modulation by a GTP-binding protein. At the moment, little is known about the molecular characteristics of the final step in the process of regulated exocytosis, i.e. the fusion of the membrane of a secretory vesicle with the plasma membrane. We have reconstituted this fusion event in vitro, using zymogen granules and plasma membranes from the exocrine pancreas of the rat. The membranes of zymogen granules were loaded with the lipid-soluble fluorescent probe octadecylrhodamine B, at a concentration that resulted in self-quenching of its fluorescence. The granules were then incubated with pancreatic plasma membranes at 37 degrees C, and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Zymogen granules fused with pancreatic plasma membranes, but not with plasma membranes from liver or chromaffin cells; granules also fused with unlabelled granule membranes. The fusion of granules with plasma membranes was unaffected by variation of the Ca2+ concentration over a wide range, but fusion of granules with both plasma membranes and zymogen granule membranes was stimulated by GTP and, more potently, by guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The effect of GTP[S] was to increase the extent of fusion occurring at low concentrations of plasma membranes, without affecting the maximum signal obtained at high membrane concentrations. Pre-incubation of the plasma membranes with GTP[S] also enhanced their ability to fuse with zymogen granules. Our results indicate that membrane fusion during exocytosis may be under the direct control of a GTP-binding protein.


Nitric oxide and cyclic GMP formation induced by interleukin 1 beta in islets of Langerhans. Evidence for an effector role of nitric oxide in islet dysfunction. Treatment of pancreatic islets with interleukin 1 (IL-1) results in a time-dependent inhibition of glucose-stimulated insulin secretion which has recently been demonstrated to be dependent on the metabolism of L-arginine to nitric oxide. In this report IL-1 beta is shown to induce the accumulation of cyclic GMP (cGMP) in a time-dependent fashion that mimics the time-dependent inhibition of insulin secretion by IL-1 beta. The accumulation of cGMP is dependent on nitric oxide synthase activity, since NG-monomethyl-L-arginine (a competitive inhibitor of nitric oxide synthase) prevents IL-1 beta-induced cGMP accumulation. cGMP formation and nitrite production induced by IL-1 beta pretreatment of islets are also blocked by the protein synthesis inhibitor, cycloheximide. The formation of cGMP does not appear to mediate the inhibitory effects of IL-1 beta on insulin secretion since a concentration of cycloheximide (1 microM) that blocks IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and nitric oxide formation does not prevent cGMP accumulation, thus dissociating the two events. By using e.p.r. spectroscopy, IL-1 beta is shown to induce the formation of a g = 2.04 iron-nitrosyl feature in islets which is prevented by cycloheximide, demonstrating the requirement of protein synthesis for IL-1 beta-induced nitric oxide formation. Iron-nitrosyl complex-formation by islets confirms that IL-1 beta induces the generation of nitric oxide by islets, and provides evidence indicating that nitric oxide mediates destruction of iron-sulphur clusters of iron-containing enzymes. Consistent with the destruction of iron-sulphur centres is the finding that pretreatment of islets with IL-1 beta results in an approx. 60% inhibition of mitochondrial oxidation of D-glucose to CO2. Inhibition of islet glucose oxidation appears to be mediated by nitric oxide since both NMMA and cycloheximide prevent IL-1 beta-induced inhibition of glucose oxidation. These results show that IL-1 beta-induced nitric oxide formation parallels the ability of IL-1 beta to inhibit glucose-stimulated insulin secretion by islets, and that protein synthesis is required for IL-1 beta-induced nitric oxide formation. These results also suggest that nitric oxide mediates IL-1 beta-induced inhibitory effects on the pancreatic beta-cell by functioning as an effector molecule responsible for the destruction of iron-sulphur centres of iron-containing proteins, resulting in an impairment of mitochondrial function.


The human hepatoma Hep3B cell line as an experimental model in the study of the long-term regulation of acute-phase proteins by cytokines. The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.


The oxidative pentose phosphate pathway in the heart: regulation, physiological significance, and clinical implications. The capacity of the oxidative pentose phosphate pathway (PPP) in the heart is small, since the activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and rate-limiting enzyme, is very low. Basically, two mechanisms are involved in the regulation of this pathway. Under normal conditions, G-6-PD is inhibited by NADPH. This can immediately be overcome in the isolated perfused rat heart by increasing the oxidized glutathione and by elevating the NADP+/NADPH ratio. Apart from this rapid control mechanism, there exists a long-term regulation which involves the synthesis of G-6-PD. All catecholamines that were administered stimulated the activity of myocardial G-6-PD in a time- and dose-dependent manner. This stimulation was due to increased new synthesis of enzyme protein, since the G-6-PDmRNA was specifically enhanced. As a consequence of the stimulation of the oxidative PPP, the available pool of 5-phosphoribosyl-1-pyrophosphate (PRPP) was elevated which serves as an important precursor substrate for purine and pyrimidine nucleotide synthesis. The limiting step in the oxidative PPP can be bypassed by ribose which leads to an elevation of the cardiac PRPP pool. The decline in the ATP that is induced in many pathophysiological conditions can be attenuated or even entirely prevented by i.v. infusion of ribose. In some experimental in vivo rat models such as in the overloaded and catecholamine-stimulated heart and in the non-ischemic region of the infarcted heart, the normalization of the metabolic situation was accompanied by an improvement of global heart function. Ribose application has been shown to be beneficial in several clinical disease states such as myoadenylate deaminase deficiency and McArdle's disease. Moreover, ribose facilitated thallium-201 redistribution and markedly improved the detection of reversible ischemic injury of the pig and human heart.


Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking. Evidence for protein kinase C-independent and -dependent mechanisms of activation. Cross-linking of membrane immunoglobulin (mIg), the B lymphocyte antigen receptor, with anti-receptor antibodies stimulates tyrosine phosphorylation of a number of proteins, including one of 42 kDa. Proteins with a similar molecular mass are tyrosine-phosphorylated in response to receptor stimulation in other cell types and have been identified as serine/threonine kinases, termed mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs). The MAP kinases constitute a family of related kinases, at least three of which have molecular masses of 40-45 kDa. In this paper we show that mIg cross-linking stimulated the myelin basic protein phosphotransferase activity characteristic of MAP kinase in both mature and immature murine B cell lines. This enzyme activity co-purified on three different columns with a 42 kDa protein that was tyrosine-phosphorylated (pp42) in response to mIg cross-linking and which reacted with a panel of anti-(MAP kinase) antibodies. Although immunoblotting with the anti-(MAP kinase) antibodies showed that these B cell lines expressed both 42 kDa and 44 kDa forms of MAP kinase, only the 42 kDa form was activated and tyrosine-phosphorylated to a significant extent. Activation of protein kinase C (PKC) with phorbol esters also resulted in selective tyrosine phosphorylation and activation of the 42 kDa MAP kinase. This suggested that mIg-induced MAP kinase activation could be due to stimulation of PKC by mIg. However, mIg-stimulated MAP kinase activation and pp42 tyrosine phosphorylation was only partially blocked by a PKC inhibitor, the staurosporine analogue Compound 3. In contrast, Compound 3 completely blocked the ability of phorbol esters to stimulate MAP kinase activity and induce tyrosine phosphorylation of pp42. Thus mIg may activate MAP kinase by both PKC-dependent and -independent mechanisms.


Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line. We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.


Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide. A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.


Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein. FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.


In vitro synthesis of bacterio-opsin: integration into microsomal membranes. The translation and membrane integration of bacterio-opsin from Halobacterium salinarium were investigated. Plasmids containing the bacterio-opsin-gene with or without its original presequence were transcribed with the T7-RNA-polymerase and translated in vitro in a wheat germ system. The integration of the expressed bacterio-opsin into dog pancreas microsomes was studied. Both precursor bacterio-opsin and mature bacterio-opsin integrate into the eukaryotic membrane.


1,10-Phenanthroline-copper, a footprinting reagent for single-stranded regions of RNAs. The 1,10-phenanthroline-cuprous complex (OP-Cu) with hydrogen peroxide as a coreactant nicks the single-stranded loops and bulges of RNA stem-loop structures more rapidly than the double-stranded stems. This chemical nuclease is therefore a useful footprinting reagent for these regions and can be used to monitor both intramolecular and intermolecular hybridization of single-stranded domains. The formation of A-form structures characteristic of either RNA-RNA or RNA-DNA duplexes inhibits scission because it blocks the binding site of the coordination complex in single-stranded loops and not because the oxidatively sensitive hydrogens of the ribose moiety are blocked. The C-4' and C-1' hydrogens are accessible to solvent in A-structures.


RNA hydrolysis via an oxyphosphorane intermediate. From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5') bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.


Cross-linking of surface immunoglobulin activates src-related tyrosine kinases in WEHI 231 cells. Crosslinking of sIgM on the B cell line WEHI 231 with anti-sIgM antibody induces protein tyrosine phosphorylation, implicating protein tyrosine kinases (PTKs) in sIg-mediated signal transduction. We have analyzed this cell line for members of the src family of PTKs and have evaluated whether these PTKs might be involved in the process of sIgM-mediated signaling. Our results show that Blk, Lyn, Lck, and Hck are detectable in WEHI 231 cells. Addition of antibodies to sIgM were found to variably stimulate the activities of Blk, Lyn, Lck, and Hck as measured by immune-complex protein kinase assays. Autophosphorylation of these src PTKs, as assessed by reaction with anti-phosphotyrosine antibodies, increased over the time course of sIgM-mediated activation. Co-immunoprecipitation studies to investigate the potential physical interaction of src PTKs with the sIgM receptor complex revealed that, under digitonin and Brij 96 lysis conditions Lyn, Lck, Hck, but not Blk associated with sIgM.


Detection of endothelin-1 mRNA by RT-PCR in isolated rat renal tubules. A combination of reverse transcription and polymerase chain reaction was utilized to detect baseline endothelin-1 (ET-1) mRNA expressed in renal tubules dissected from rats. 5' and 3' primers were constructed according to human ET-1 genomic DNA. Rat ET-1 mRNA was found to be expressed only in cortical through medullary collecting ducts in addition to glomeruli. Sites for tubular synthesis of ET-1 corroborate well with major ET-1 binding sites along the nephron, indicating autocrine/paracrine role of ET-1 in the renal tubules and supporting a prevailing concept on such function of ET-1 in many differing tissues.


A serine-->proline change in the Alzheimer's disease-associated epitope Tau 2 results in altered secondary structure, but phosphorylation overcomes the conformational gap. Monoclonal antibody Tau 2 was raised against bovine tau protein, was reported to recognize a conformational epitope, and stained tau was found in neurofibrillary tangles of Alzheimer's disease, but not normal human tau. We synthesized tetradeka peptides corresponding to the original bovine sequence, its serine-->proline substituted analog, the genuine human sequence of this region, and the bovine epitope phosphorylated on the crucial serine. The secondary structure of the peptides was determined by circular dichroism. It was found that only the original bovine epitope showed a tendency to form the beta-pleated sheets characteristic of the neurofibrillary tangles. The spectra of the human peptide, its analog, and the phosphorylated bovine sequence were very similar, featuring a weak, helical beta-turn character. Eventual phosphorylation of epitopes of this otherwise heavily phosphorylated protein may overcome inter-species conformational gaps.


Analysis of antibody response to ovine lentivirus by using viral gene products expressed in a prokaryotic system. The polymerase chain reaction (PCR) was used to amplify, clone, and express eight DNA fragments encoding p25, p16, reverse transcriptase (RT) core, C'-terminal RT, N'- and C'-terminals of external (gp70), and transmembrane (gp40) envelope proteins from visna virus infectious recombinant DNA. Efforts were focused on characterizing the nature of the humoral immune response of ovine progressive pneumonia (OPP) virus infected animals and identifying the conserved and prime-reactive antigenic determinants that have potential diagnostic value. This communication reports that the N'-terminal region of gp40 appeared to be the most immunoreactive of the bacterially expressed proteins and could serve as a sensitive immunodiagnostic antigen for the detection of OPP infection.


Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes. Incubation of rabbit articular chondrocytes with interleukin-1 beta caused time-dependent expression of NO synthase, determined as nitrite, after a lag period of 6h. The synthesis of nitrite was concentration-dependent and was inhibited by cycloheximide and NG-monomethyl-L-arginine, but not by dexamethasone or hydrocortisone. The synthesis of NO in the 100,000g supernatant of activated chondrocytes was inhibited by EGTA, but not by the calmodulin inhibitors W-13 or trifluoperazine. The synthesis of NO was half-maximal at approximately 20nM free Ca2+. Endotoxin also induced the expression of this NO synthase. Thus, rabbit articular chondrocytes express a novel inducible NO synthase which is Ca(2+)-dependent, and whose induction is not prevented by glucocorticoids.