Controversies in the diagnosis and management of benign prostatic hypertrophy. The prevalence of BPH ensures a continuing need for internists to be aware of the issues regarding the evaluation and treatment of prostatism, while the developing role of nonoperative therapy and the growing recognition of the importance of patient preferences have opened the way for an expanded involvement of the primary care practitioner in the management of these patients. Prostatism remains a difficult clinical problem since many of the symptoms and diagnostic tests are nonspecific, especially in the elderly. Basic questions regarding the role of bladder outlet obstruction (BOO) in the development of symptoms and in predicting outcome from surgery await further study. Against this background of uncertainty, however, is the reassurance that the natural history of the process results in serious complications in only the minority of patients. Important work is under way that will incorporate quality of life outcomes, compare surgery with "watchful waiting," and randomize patients to surgical and nonsurgical treatments. In the interim, the internist may help patients with prostatism significantly by appreciating the natural history of the condition, treating the conditions that mimic or exacerbate it, understanding the clinical utility and limitations of current evaluation methods, becoming aware of the growing array of nonsurgical and minimal-surgery options, and assisting patients in weighing therapeutic risks and benefits with their concerns about symptoms.


[Laser photocoagulation for experimental choroidal neovascularization. 1. Mild dye laser photocoagulation]. The author treated experimentally produced choroidal neovascularization (ChNV) with mild dye laser photocoagulation (PHC). We treated these ChNVs with 590 nm wavelength, 200 microns spot size, 0.2 second duration and 50 mW of power. Eleven eyes of nine rhesus monkeys were used. Then these ChNVs were examined clinically and histopathologically at 24 hours, 2 weeks and a month after therapeutic PHC. Small ChNVs, less than 1/3 disc diameter healed successfully and histopathologically most of them were coagulated and disappeared. A few poorly developed ChNVs remained, but were well enveloped by the proliferating retinal pigment epithelial (RPE) cells. On the other hand, ChNVs of more than 1/3 disc diameter did not respond to weak PHC, grew actively over a month, and were not completely surrounded by proliferating RPE cells. These results suggest that by mild PHC, small ChNVs may be treatable, however, in large ChNVs, mild PHC may rather accelerate their activity.


Infant neuromotor assessments: a review and preview of selected instruments. The advancement of medical technology has produced an increased number of surviving neonates. Occupational therapists and physical therapists working in neonatal intensive care units and follow-up clinics are faced with the challenge of providing early assessment and treatment for this relatively new population of infants. The purpose of this article is to review selected infant motor evaluation instruments used by therapists and to preview those currently being developed. An overview of the historical background of infant neuromotor testing is provided, followed by a review of the Infant Neurological International Battery (Ellison, Horn, & Browning, 1985) and the Neonatal Neurobehavioral Examination (Morgan, Koch, Lee, & Aldag, 1988). Two infant motor tests currently being developed, the Chandler Movement Assessment of Infants Screening Test and the Miller Infant and Toddler Test (both of which, at the time of this writing, are unpublished) are described. Updated information on the psychometric properties and the clinical usefulness of these new infant neuromotor tests can assist therapists in selecting reliable and valid measures in their clinical practice.


Efficacy and efficiency: self-designed versus instructor-designed study tools. During the course of their education, occupational therapy students learn to administer complex structured assessments. For easier administration of these assessments, students design note cards, which then replace cumbersome test manuals during administration. This study considered whether students could learn test administration with equal efficiency and efficacy if given test administration note cards rather than having to design their own. The results showed that the subjects using instructor-designed cards earned written test and practical examination scores similar to those of the subjects using self-designed cards. The subjects using instructor-designed cards spent significantly less (p = .003) total time in study than did the subjects using self-designed cards. The difference in time between the two groups was attributable to the time spent designing note cards. Therefore, distribution of instructor-designed note cards appears to offer equally effective and significantly more efficient learning when compared with that produced when students design their own cards. The differences in efficacy and efficiency were similar for students of different learning styles (as classified by Witkin's field-dependence/field-independence continuum) [corrected].


Should music be used therapeutically in occupational therapy? It has long been common knowledge that music profoundly affects human beings on a variety of levels. Occupational therapists have at their disposal a potentially powerful therapeutic tool, but the specific effects of this tool have not been documented. Occupational therapists have a history of using music in their treatment, probably to a greater extent than is documented in the literature. It would be advantageous for us to engage in research regarding the application of music to occupational therapy.


Gene expression of growth-related proteins and ECM constituents in response to unilateral nephrectomy. To identify the specific regulatory mechanism associated with the events following unilateral nephrectomy, we measured the levels of mRNA encoding for extracellular matrix (ECM) constituents, for protooncogenes, and for proliferating cell nuclear antigen (PCNA) in renal cortex and glomeruli. One hour after left nephrectomy, c-jun and c-fos mRNA levels in renal cortex increased rapidly and then decreased rapidly to the control level, whereas c-myc and PCNA mRNA levels showed a slower and more sustained increase, with a peak at 6 h after nephrectomy, and then decreased to the control level after 7 days. mRNA levels for basement membrane components including alpha 1-chain of type IV collagen, laminin B1 and B2 chains, and heparan sulfate proteoglycan core protein were significantly increased in renal cortex at 12 h after nephrectomy, whereas those for interstitial collagens including alpha 1-chains of type I and type III collagen were unchanged following nephrectomy. On the other hand, the glomerular expression of all genes examined in this study showed little change during the experimental period. These results suggest that the time course of mRNA expression of ECM constituents is different from that of growth-related proteins in renal cortex and that glomerular mRNA levels for these components may not be associated with renal hypertrophy in the early stages following unilateral nephrectomy.


Use of blue dextran for measuring changes in perfused vascular surface area in lungs. We investigated the uptake and efflux of Blue Dextran in the isolated perfused rabbit lung. Blue Dextran is a high-molecular-weight glucose polymer (original mol wt 2 x 10(6) g/mol) containing covalently bonded Reactive Blue 2 dye (approximately mmol/g dextran). This blue dye is known for its high binding affinity to a wide variety of proteins, with a particularly high affinity for serum albumin. In isolated rabbit lungs perfused with a protein-free perfusate, both bolus injection and recirculation of Blue Dextran revealed a rapid saturable uptake. Once the lungs were loaded with Blue Dextran, efflux of the Blue Dextran accumulated in the lungs could be induced by addition of bovine serum albumin (BSA) to the recirculating perfusate. The amount of BSA-induced efflux of Blue Dextran from the lung was independent of perfusate flow. When the left pulmonary artery was ligated after the lungs had been loaded with Blue Dextran, the dye-induced BSA efflux was only about 50% of normal. Release of the ligature so that both lungs were perfused resulted in efflux of the remaining Blue Dextran. The combination of high airway pressure and low flow also reduced the dye efflux, and the effect was reversed by reducing the airway pressure. With the assumption that the high average molecular weight of Blue Dextran confines this molecule to interaction with proteins on the vascular surface, the results of this study suggest that Blue Dextran uptake and its BSA-induced efflux are proportional to the perfused vascular surface area in the lung.


Cyclic nucleotide phosphodiesterases from frog atrial fibers: isolation and drug sensitivities. The cyclic nucleotide phosphodiesterase (PDE) forms present in frog atrial fibers were isolated and characterized by their drug sensitivities. DEAE-sephacel chromatography of cytosolic PDE activity resolved three major PDE forms: peak A hydrolyzed both adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and was activated by calcium-calmodulin (PDE I); peak B also hydrolyzed both cAMP and cGMP but was activated by 5 microM cGMP (PDE II); peak C specifically hydrolyzed cAMP (PDE IV). Rolipram specifically inhibited PDE IV (Ki = 1.1 microM), whereas dipyridamole potently inhibited both PDE II (Ki = 4.6 microM) and PDE IV (Ki = 0.8 microM). Atrial fiber PDE I was preferentially inhibited by zaprinast (Ki = 10 microM). 3-Isobutyl-1-methyl xanthine (IBMX) and theophylline inhibited nonspecifically all three different enzymes. The positive inotropic drug CI 930 only inhibited the different isolated atrial PDE forms at concentrations greater than 200 microM. However, under assay conditions for which PDE IV was specifically inhibited (presence of 100 microM rolipram), an IC50 of 17 microM for CI 930 was observed on the remaining 26% cAMP hydrolytic activity of peak C (which could represent a cGMP-inhibited PDE form: PDE III). The same PDE forms were also found in frog ventricle. The major difference between frog atrial fiber (and ventricular tissue) PDEs and mammalian cardiac PDEs is that the main cytosolic cAMP-specific hydrolytic activity in frog heart is due to PDE IV rather than PDE III. Rolipram, dipyridamole, and zaprinast might be useful tools to investigate the participation of cAMP in frog atrial contraction (unpublished observations).


Systemic and regional O2 delivery and uptake in bled dogs given hypertonic saline, whole blood, or dextran. The mechanisms by which small volumes of hypertonic saline in dextran (HSD) resuscitate bled dogs are incompletely understood but may include a pulmonary osmolar reflex. A known negative effect of HSD is hemodilution that reduces O2-carrying capacity. Our goals in this study were to ascertain whether the putative osmotic reflex redistributed blood flow between muscle and gut and whether O2 delivery (DO2) was adequate at systemic and regional levels. Left hindlimb muscle and a segment of ileum were vascularly isolated in three groups (n = 8) of anesthetized dogs that were then bled to mean arterial pressure (MAP) of 40 mmHg for 30 min. At that point, all shed blood (approximately 40 ml/kg) was returned in the blood group (BLD); 20 ml/kg of Dextran 70 was given to the dextran group (DEX); and 5 ml/kg of 7.5% NaCl in dextran was given to the HSD group. MAP and cardiac output were restored to acceptable levels in all but was poorly maintained in HSD. The fall in hematocrit (41 to 25%) in HSD was matched by that in DEX (42 to 22%), so that DO2 only reached approximately 55% of that in BLD. Nevertheless, systemic and regional O2 uptakes were similar; O2 debt and repayment did not differ; and lactate metabolism was alike in all groups. O2 extraction did have to increase to near maximum in HSD, however. Other than a transient increase to muscle, HSD had no special effect on distribution of cardiac output. HSD was efficacious as a short-term resuscitative measure but did encroach markedly on O2 transport reserves.


Gene expression in skeletal muscle in response to stretch and force generation. Striated muscle is a tissue in which gene expression is influenced to a large extent by mechanical signals. This includes the regulation of gene expression-associated muscle fiber phenotype determination, which depends on which protein isoform genes are transcribed, as well as muscle fiber mass accretion, which appears to involve some translational regulation. Although muscle synthesizes a set of highly specialized proteins it has a remarkable ability to adapt by expressing different isoforms of the same protein so that it acquires the appropriate contractile characteristics. Our work has focused on the myosin heavy chain (HC) genes as these encode the myosin cross bridge, which is responsible for muscle intrinsic velocity of contraction and economy of force development. RNA analyses after cast immobilization of the limb with the muscle in the lengthened or shortened position and/or with electrical stimulation were used to determine the effects of altered mechanical signals on gene transcription. When the soleus muscle was immobilized in the shortened position in the young animal it did not fully differentiate into a slow postural-type muscle. Even in the adult, the soleus muscle if deprived of stretch and contractile activity switches back to transcribing the fast myosin HC gene. The converse was true when the fast rabbit tibialis anterior was subjected to immobilization in the lengthened position and/or electrical stimulation. Both stretch alone and stimulation alone caused repression of the fast type and activation of the slow myosin genes. The reprogramming of the fast muscle was more complete when the stretch was combined with stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Insulin-like growth factor I preserves host lean tissue mass in cancer cachexia. Insulin-like growth factor I (IGF-I) has been implicated in the regulation and maintenance of skeletal muscle protein balance and thus may be of potential benefit in attenuating the cancer-cachectic process. To examine this hypothesis, 47 sham or tumor-implanted Fischer 344 rats were randomized to receive either continuous subcutaneous IGF-I (220 or 400 micrograms/day) or saline as control. In the tumor-bearing (TB) population, IGF-I-treated groups showed a dose-dependent increase in host weight gain (P less than 0.05), final carcass weight (P less than 0.05), and gastrocnemius muscle weights (P less than 0.05) and protein contents (0.50 +/- 0.02, 0.40 +/- 0.01, and 0.52 +/- 0.03 g/100 g host wt, for non-TB saline, TB saline, and TB 400 mg IGF-I groups, respectively; P less than 0.01, IGF-I vs. saline). Similar increases in muscle RNA and DNA contents (P less than 0.01) were induced by IGF-I treatment (P less than 0.05). IGF-I treatment in this rat sarcoma model significantly reduced the proportion of aneuploid cells in the tumor (aneuploid-to-diploid ratio: TB saline 1.1 +/- 0.2 vs. TB IGF-I 0.5 +/- 0.1; P less than 0.05). IGF-I treatment attenuated host muscle protein and lean tissue depletion without stimulation of tumor growth. The tumor aneuploid population was reduced in response to IGF-I treatment. Thus IGF-I may be a potential therapeutic agent in cancer-induced cachexia.


Renal nerves modulate kidney renin gene expression during the transition from fetal to newborn life. The role of renal nerves in regulating changes in plasma renin activity (PRA) and renal renin gene expression was studied in intact (n = 6) and denervated (n = 6) fetal sheep before birth and during the first 24 h after delivery. Renal denervation completely blunted the rise in PRA observed 24 h after delivery in newborn lambs; in lambs with intact kidneys, PRA increased significantly (P less than 0.05) from 3.26 +/- 0.60 (predelivery) to 6.34 +/- 1.85 ng angiotensin I (ANG I).ml-1.h-1 (24 h postdelivery), while in lambs with denervated kidneys, predelivery and post-delivery values were 2.84 +/- 0.19 and 2.49 +/- 0.45 ng ANG I.ml-1.h-1, respectively. Renin mRNA levels were significantly lower (P less than 0.001) in denervated than in intact kidneys 24 h after birth. A close analysis of these results also revealed that renin mRNA levels were significantly higher (P less than 0.001) in intact kidneys of newborn lambs delivered vaginally (n = 3) than in newborn lambs delivered by cesarean section (n = 3). These results suggest that renal nerves play an important role in regulating renin gene expression and PRA during the transition from fetal to newborn life.


Antibody response to the circumsporozoite protein of Plasmodium vivax in naturally infected humans. The circumsporozoite (CS) protein of Plasmodium vivax consists of a central repeat region flanked by highly conserved non-repeat regions. Serum samples from 33 individuals with naturally acquired infections of P. vivax were tested for antibodies to four antigens representing the vivax CS protein. Three recombinant proteins containing different overlapping sequences in the non-repeat regions and either the entire central repeat region (vivax-1 and vivax-2) or two of the repeat sequences (vivax-3) were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Antibodies to two other proteins, one (NS1(81)V20) containing the entire predominant repeat region (GDRAA/DGQPA) and the other (Pvk247) containing the variant repeat sequence (ANGAGNQPG) that was recently reported from Thailand were also measured by ELISA. Immunoglobulin G antibodies to the antigen representing the predominant repeat were present in 15% of the patients on the first day of treatment (day 0) and in 24% of the patients two weeks later (post-treatment). Six and 12% of the patients had IgG antibodies to the antigen containing the variant repeat on day 0 and post-treatment, respectively. A larger proportion of the sera had antibodies to the three antigens containing the non-repeat sequences; on the first day of treatment and two weeks later, 79 and 97% of the patients, respectively, had antibodies to vivax-1, vivax-2, and vivax-3. In this sample of Peruvians naturally infected with P. vivax, the most prevalent antibody responses were targeted to epitopes in the non-repeat region of the CS protein rather than to epitopes in the repeat region.(ABSTRACT TRUNCATED AT 250 WORDS)


Sacral resection for control of pelvic tumors. A surgical approach for treating patients with resected, recurrent, posterior pelvic visceral tumors involving the sacrum is detailed. Of 11 patients, 9 had rectal cancers, 1 had chordoma, and 1 had cancer of the cervix. Five total pelvic exenterations and five posterior exenterations were performed en bloc with involved sacrum. One patient had a sacral resection only. Surgical mortality was 9%, and the average hospital stay was 1 month. Mean disease-free survival was 1 year, and mean survival was 3 years. Absolute cure rate was 18% with a complete 5-year follow-up. This experience confirms the value of this procedure in selected patients.


Primary treatment of hepatocellular carcinoma by arterial chemoembolization. Two hundred and ninety-one patients with hepatocellular carcinoma were treated by chemoembolization (CE), using ethiodized oil, doxorubicin, and a gelatin sponge. Patients with thrombosis of either the portal vein or a main branch were excluded. The mortality rate in the first 2 months after treatment was 7% in noncirrhotic patients, 2.8% in patients with class A cirrhosis, 8% in patients with class B cirrhosis, and 37% in patients with class C cirrhosis. The tumor diameter remained the same in 55.3% of patients, was reduced by up to 50% in 20% of the patients, was reduced by more than 50% in 7.3% of the patients, and almost completely disappeared in 1.8% of the patients. The diameter of the tumor increased in 15.6% of patients. Forty-three patients underwent a resection or transplantation after chemoembolization. Histologic examination of the specimens revealed significant necrosis of the tumor. The long-term survival rate at 2 years was 49% for class A cirrhotics, 29% for class B cirrhotics, and 9% for class C cirrhotics. Complications included cholecystitis (10%), vasculitis (14%), renal decompensation (13%), an increase in ascites (14%), and jaundice (12%). Chemoembolization is an effective and safe initial treatment for hepatocellular carcinoma. It is effective in producing tumor necrosis and reducing the size of the tumor. Improvement in survival was noted when patients who underwent chemoembolization were compared with an historical series of untreated patients, and resection and transplantation are kept as options.


Direct measurement of fast axonal organelle transport in chronic ethanol-fed rats. While indirect methods have been used, direct evaluation and measurement of the fast axonal transport system itself in chronic ethanol-fed rats have not been carried out previously. We evaluated this system using analog and digital image enhancement of differential interference contrast optical images in real time to assess the effect of ethanol on fast intra-axonal organelle traffic in rat sural nerve. A radiolabeling method for evaluating fast axonal transport was used in similar animals to compare the indirect and direct techniques. Also, the concentration of organelles (mitochondria, large and small clear vesicles, dense vesicles, and membranous whorls) in the annulospiral sensory nerve endings of muscle spindles from a foot muscle (flexor digitorum brevis) of these same animals was quantitated from electron micrographs. Rats were fed an ethanol-containing liquid diet for one to five months. Three observations were made; (1) There was no statistically significant change in the mean organelle speed in the anterograde direction, but the mean organelle speed in the retrograde direction increased 11%, 9%, and 17% (statistically significant) at 3, 4, and 5 months of ethanol exposure, respectively. (2) Significant increases in organelle content of sensory nerve endings were seen at 2 and 3 months of intoxication. (3) Increases in organelle densities in terminals were transitory and returned to normal in the face of on-going ethanol administration. We conclude: (1) There is no permanent impairment of fast axonal organelle transport in this model after 5 months of exposure. (2) Sensory endings on muscle spindles show transitory increases in organelle density. (3) Retrograde speed increases may be a partial compensatory mechanism to help restore normal terminal organelle density.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of histamine antagonists and agonists on IgE production in mice. This study was undertaken to investigate the effect of histamine, its receptor antagonists and agonists on IgE antibody production in mice. BALB/c mice were immunized intraperitoneally with 1 mg alum plus 30 micrograms of Ag90, the antigen of Japanese occupational asthma. Histamine receptor antagonists were administered before the immunization and simultaneous injection of histamine. The mice were bled 14 days after immunization and anti-Ag90 IgE antibody was obtained. Titers of the antisera were measured by passive cutaneous anaphylaxis reaction in Sprague-Dawley rats. Treatment with histamine only did not affect the level of specific IgE antibody. Administration of H1 antagonist or H2 agonist suppressed IgE production significantly. In contrast, treatment with H2 antagonist or H1 agonist augmented the IgE antibody production. Injection of H1 + H2 antagonists had no effect on the antibody production. These results suggest that histamine suppressed specific IgE production via H2 receptors and enhanced it through H1 receptors in the induction phase of the system.


Relationship between allergen-specific skin testing and nasal provocation in patients with perennial rhinitis. Patients with perennial rhinitis were evaluated by allergen challenge epicutaneously and intranasally. Nasal provocation was assessed by clinical score, graded 0-12, to include rhinomanometry, secretions (mL), sneezes, and stuffy nose. All 40 patients with perennial symptoms had no clinical history of seasonal exacerbations but did have both positive skin tests and nasal provocation to the same allergen. Skin test wheel size correlation with nasal provocation (P less than .02).


[A case of verrucous carcinoma of the tongue, effectively treated with preoperative chemotherapy (UFT, CDDP, PEP) and irradiation]. A 70-year-old male complained of a mass on the left side of his tongue. A biopsy specimen was diagnosed as verrucous carcinoma. He was treated with preoperative chemotherapy (UFT, CDDP, PEP) and irradiation, continuous partial glossectomy. The tumor response to chemotherapy was excellent, and no tumor cells were found in the surgical specimen. There was no recurrence of the tumor 1 year and 7 months after therapy. For advanced tumors, preoperative chemotherapy appears to be significantly effective.


[A case of locally advanced esophageal cancer effectively treated with concurrent chemotherapy and radiotherapy]. A case of a 69-year-old man with locally advanced esophageal cancer was reported. The patient received five courses of chemotherapy consisting of cisplatin (30 mg i.v. days 1-5), 5-fluorouracil (500 mg i.v. days 1-5) and bleomycin (5 mg i.m. days 1-5) every four weeks with a split course irradiation at a dose of 50 Gy, which was concurrently given at the second, third and fourth course of the chemotherapy. Treatment was effectively carried out without severe toxicities. The patient achieved a histologically-confirmed complete remission after completion of the treatment. The patient is disease-free and fully active 28 months after the beginning of chemotherapy. The treatment modality appears to be useful for advanced esophageal cancer.


[Successfully treated locally advanced breast cancer by intra-arterial infusion chemotherapy: a case report]. A 40-year-old female was admitted to our hospital with a large right breast tumor that was over 15 cm in diameter. We treated this locally advanced breast cancer by intra-arterial infusion chemotherapy. Through a catheter placed in the right subclavian artery, doses of 20-30 mg of ADM were injected intermittently with MMC and 5-FU. When a total of 120 mg of ADM had been infused, leukopenia developed, but this was immediately improved by G-CSF. With this treatment, her breast tumor and lung metastases were almost completely disappeared. Thus, an intra-arterial infusion chemotherapy was considered to be an effective treatment for locally advanced breast cancer.


Biochemical changes in kidneys of normal and stone forming rats with sodium pentosan polysulphate. The influence of sodium pentosan polysulphate was studied on the deposition of stone forming constituents along with certain enzymes in the renal tissue of experimentally induced urolithiatic rats. Calcium, oxalate and phosphorus levels were elevated in kidneys of lithogenic rats, while SPP administration reduced these levels to near control values. The elevation in kidney LDH was significant in the stone forming groups and SPP had minimal effect. Increases in the activities of Na+, K(+)-and Ca(2+)-ATPases in the calculogenic groups was lowered considerably with SPP treatment. Inorganic pyrophosphatase activity was reduced significantly in the calculogenic as well as in the drug treated groups. Leucine aminopeptidase was decreased in the calculogenic group. SPP treatment elevated the enzyme activity in the treated groups. Reduction in kidney oxalate with SPP may prove useful in the medical management of urolithiasis.


Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears. Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).


Influence of cell cycle synchronization on digital image analysis of HL-60 granulopoiesis. Retinoic acid (RA)-treated HL-60 cells subjected to density arrest (DA) and double thymidine block (TB) synchronization demonstrated image feature changes associated with cellular proliferation and differentiation. RA-treated TB cells demonstrated an increased level of morphologic differentiation (assessed by differential counts and quantitation of nuclear shape) and more rapid functional differentiation (assessed by superoxide production and expression of complement receptors) than RA-treated DA cells. By comparison to DA cells, TB cells had less variation in virtually all image features values. A Kruskal-Wallis test of image features ranked total optical density (TOD) of Feulgen-stained cells, nuclear area and shape factor as the top three features regardless of synchronization method. Statistically significant changes in image feature values of RA-treated cells were first noted on day 1. The computer-assisted ability to discriminate RA-treated cells on a given day after induction from paired controls by means of an unsupervised learning algorithm increased over a seven-day period for both DA and TB cells. However, in the dichotomous (RA-treated versus untreated) classification scheme employed, which did not account for continuous levels of morphologic differentiation, there was no advantage in the use of the TB over DA procedure.


Double reactivity of monoclonal and polyclonal rheumatoid factors for IgG and histones: mapping of binding sites by means of histone synthetic peptides and anti-Id antibodies. Polyreactive antibodies able to bind various apparently unrelated structures represent a frequent antibody population in autoimmune diseases. In this work, the structural basis of the double reactivity of such autoantibodies was investigated using as models polyclonal and monoclonal human rheumatoid factors (RF) reacting with histones. Both direct ELISA binding and competitive inhibition experiments were performed. A more precise delineation of the histone regions recognized by the RFs was made by means of 27 synthetic peptides of these proteins. Anti-idiotope (Id) murine antibodies were used to map the binding sites involved on RF in the interaction with IgG and histones. Among the 13 polyclonal and six monoclonal RFs tested, four and two respectively were found to cross-react with IgG and histones. The fragments shown to be the most frequently recognized by RFs were located in residues 1-16 and 204-218 of H1, 1-20 and 65-85 of H2A, and 1-21 of H3. The results obtained by competitive ELISA assays using IgG, histone peptides and anti-Id monoclonal antibodies led us to confirm and characterize more precisely our previous finding suggesting the existence of topographically distinct binding sites for the different targets recognized by RFs.


Induction of intercellular adhesion molecule-1 but not of lymphocyte function-associated antigen-3 in thyroid follicular cells. We investigated the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) by thyrocytes and their regulation by cytokines. Immunofluorescence studies on cryostat sections and on freshly dispersed cell preparations showed that ICAM-1 and LFA-3 are barely detectable in non-autoimmune thyrocytes. However, thyrocytes acquired ICAM-1 expression in culture. IFN-gamma, IL-1 beta and TNF-alpha produced a clear enhancement of ICAM-1 expression. When tested in combination, IL-1 beta and TNF-alpha were additive to the IFN-gamma effect. LFA-3 expression was not modulated by these cytokines. In the HT93 thyroid cell line generated by transfection with SV40, ICAM-1 and LFA-3 were both constitutively expressed at high levels. Cytokines modulated ICAM-1 expression similarly, but to a greater extent than in normal thyrocytes. LFA-3 remained unmodified. These results support the notion that normal thyrocytes are immunologically silent cells. The capability of cytokines to induce ICAM-1 together with HLA class I and class II-expression on thyrocytes suggests that under their influence, these cells may express all the surface molecules required for antigen presentation and/or for being recognized as target cells in the context of thyroid autoimmune disease.


A common epitope on human tumor necrosis factor alpha and the autoantigen 'S-antigen/arrestin' induces TNF-alpha production. A common epitope on S-antigen (arrestin), a potent autoantigen inducing experimental autoimmune uveoretinitis (EAU), and on human tumor necrosis factor alpha (hTNF alpha) was revealed using two monoclonal antibodies to S-antigen which inhibit EAU induction. The minimal common sequence for monoclonal antibody recognition is GVxLxD in the S-antigen/hTNF alpha amino acid sequences. Peptides containing this sequence motif exhibited monocyte activating capacity similar to the autocrine stimulatory capacity of hTNF alpha itself. In the S-antigen this activity was located from residue 40 to 50, corresponding to the peptide PVDGVVLVDPE (epitope S2). In hTNF alpha, the monocyte activating capacity correlated to residue 31 to 53, corresponding to the peptide RRANALLANGVELRDNQLVVPSE (peptide RRAN). The identified regions define common functional structures in the autoantigen and in the hTNF alpha molecule. The data suggest a regulatory function of this particular structure in TNF alpha expression and in autoimmunity.


Alkyllysophospholipid prevents induction of experimental allergic encephalomyelitis. Alkyllysophospholipids are synthetic analogues of natural phospholipids possessing a high immunomodulating and antitumoral capacity. Experimental autoimmune encephalomyelitis is a model disease for multiple sclerosis which can be induced by injecting rats with myelin basic protein, MBP. The effect of one alkyllysophospholipid, ET-18-OCH3, on the course of experimental autoimmune encephalomyelitis was investigated. It was found that animals treated with ET-18-OCH3 showed only weak signs of disease. MBP specific T-cell lines were co-cultivated with ET-18-OCH3. The compound suppressed T-cell proliferation markedly, suggesting that this might be its mode of action in vivo. Since ET-18-OCH3 has only low toxicity in man, it could be of interest to perform further studies on its effects on autoimmune, demyelinating disease.


T helper function of CD4+ cells specific for defined epitopes on the acetylcholine receptor in congenic mouse strains. We previously identified sequence segments of Torpedo acetylcholine receptor (TAChR) alpha subunit recognized by CD4+ cells of congenic mouse strains of different H-2 haplotypes, susceptible to experimental autoimmune myasthenia gravis. CD4+ cells from BALB/c and CB17 mice (H-2d) recognized the peptide sequences alpha 1-20 and alpha 304-322, while C57BL/6 and BALB/b mice (H-2b) recognized alpha 150-169 and alpha 360-378. C57BL/6 mice recognized to a lesser extent also peptide alpha 181-200. In the present study we demonstrate that CD4+ cells which recognize these epitopes have T-helper function. CD4+ cells from TAChR immunized mice, stimulated in vitro with synthetic epitope peptides, induced proliferation in vitro of B cells via soluble factors which were not strain specific, and induced secretion in vitro of anti-AChR antibodies. Upon in vitro stimulation with T-epitope peptides, they secreted interleukin-2. Immunization of mice with synthetic T-epitope peptides caused sensitization of CD4+ cells, which responded in vitro both to the immunizing peptides and to TAChR, and appearance of anti-AChR antibodies in vivo, further identifying the epitope-specific CD4+ cells as AChR-specific T-helper cells.


Four distinct antigenic regions are present in the primary structure of HIV-1 and HIV-2 proteinases. The purpose of this study was to assay reactivity of antibody-positive sera to different parts of the HIV-1 and HIV-2 proteinase (PR) proteins. Since the majority of HIV-1-antibody-positive sera react to the proteinase, but the antigenic determinants on the protein have not been identified, we attempted to identify these determinants. We synthesized 18 peptides representing the PR of HIV-1 and HIV-2 in order to map serum reactivity to the PR protein. Both HIV-1- and HIV-2-antibody-positive sera recognized four distinct antigenic regions in the HIV-1 and HIV-2 PR. Correlation between our results and the crystallographic structure of the protein revealed that the antigenic regions are positioned at the surface of the HIV-1 PR. Although the structure of HIV-2 PR has not yet been characterized, our results indicate that the folding of the HIV-1 and HIV-2 PR may be very similar.


Effect of isoprinosine on HIV antigenaemia. The objective of this study was to evaluate the effect of isoprinosine on HIV-antigen expression in HIV-positive patients without AIDS. Serum samples from anti-HIV-positive patients without AIDS participating in a double-blind, placebo-controlled trial of isoprinosine in the treatment of HIV infection were analysed for the presence of HIV antigen. Data and samples were collected from the 21 medical centres who participated in the Scandinavian multicentre placebo-controlled isoprinosine study. Samples were available from 19 of 21 participating centres. Of 866 patients who enrolled, baseline serum samples were available for 642 (74%; 308 isoprinosine- and 334 placebo-treated patients). Treatment was 1 g isoprinosine administered orally three times a day or matching placebo for 24 weeks. Comparison of HIV-antigen levels before and during treatment in both the isoprinosine-treated group and the placebo-treated group of patients. During the study, AIDS developed in 19 patients; 17 of whom were receiving placebo treatment and two isoprinosine. The proportion of HIV-antigen-positive patients developing AIDS during treatment was significantly different from the proportion of HIV-antigen-negative patients in whom AIDS developed (6 versus 2%; P = 0.02). No significant changes in HIV-antigen levels were observed between the isoprinosine- and the placebo-treated group of HIV-antigen-positive patients. Median HIV-antigen levels did not change significantly in either the isoprinosine- or the placebo-treated group. Our results suggest that isoprinosine does not have antiviral activity against HIV in vivo.


The bleomycin resistance gene of transposon Tn5 is an excellent marker for transformation of corynebacteria. Corynebacteria are highly sensitive to the glycopeptide antibiotic bleomycin. The bleomycin resistance gene of transposon Tn5 is expressed very efficiently in Brevibacterium lactofermentum. This gene constitutes an excellent marker for selection of transformants of corynebacteria. The bleomycin resistance gene is expressed from the same promoter as the neomycin resistance gene, which is already used as marker in many vectors of corynebacteria. The promoter of the neo-ble cluster is expressed in a variety of Gram-negative and Gram-positive microorganisms and eucaryotic organisms.


Intermediate filament molecular biology. Epidermal keratin intermediate filaments appear to have a structural function. The functions of other intermediate filaments are being elucidated using a combination of molecular genetic methods, including the expression of dominant negative mutant proteins and gene targeting. The differential expression of intermediate filament genes is regulated by both the accessibility of multiple regulatory elements and the activity or level of multiple positive and negative transcription factors.


Slow axonal transport.

Slow axonal transport. New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.


Intermediate filament structure.

Intermediate filament structure. In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.


Transcriptional down-regulation of c-myc in human prostate carcinoma cells by the synthetic androgen mibolerone. The mechanism of down-regulation of c-myc RNA associated with androgen-induced suppression of the transformed phenotype in the human prostate carcinoma cell line LNCaP was investigated. The synthetic androgen mibolerone (7 alpha-17 alpha-Dimethyl-19-nortestosterone) reversibly inhibits the proliferation of LNCaP cells and, from 12-72 h after hormone addition reduces the level of c-myc transcripts to a few per cent of controls. P1, P2, and P0 c-myc transcripts decline at the same rate, whereas P3 transcripts are much less hormone sensitive. Nuclear run-on analysis revealed that c-myc is down-regulated at the level of transcription initiation in LNCaP cells. The level of c-myc transcripts prevailing in untreated control cells can be restored in androgen-induced cells by excess antiandrogen, indicating the involvement of the androgen receptor in c-myc down-regulation.


In vitro effects of substance P analogue [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P on human tumour and normal cell growth. Analogues of the neurotransmitter substance P (SP) can interact with neuropeptide receptors, and are reported to inhibit growth of small cell lung cancer cell lines (SCLC CLs). We found [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P (D-Phe5SP) significantly inhibited DNA synthesis by 10/10 human tumour CLs; six SCLC, one N-SCLC (squamous), two ovarian and one squamous cervical carcinoma, with inhibition to 50% control levels (IC50) of 20-50 microM. There was dose dependent inhibition of colony forming efficiency (CFE) in 3/3 SCLC and 1/1 N-SCLC CL, IC50s of 0.5-6.5 microM in 5% serum. Exposure of SCLC CL HC12 to 100 microM D-Phe5SP for 1-4 h caused a progressive fall in viable cell number; surviving cells, grown in the absence of peptide, showed a decreased growth rate. During 1 week's exposure of two SCLC CLs to 20 microM D-Ph5SP, growth was slower than control cultures, while 50-100 microM completely inhibited growth. These inhibitory effects were partially reversed by increasing serum concentration from 5 to 20%, but not by SP, vasopressin, bombesin or insulin-like growth factor 1. There was some inhibition of CFE by 3/3 normal human bone marrows, IC50s of 30-80 microM, compared with 8 microM for HC12 in 20% FCS. Therefore D-Phe5SP appears to have more potent antiproliferative effects in tumour cells than normal cells, suggesting a role for this analogue in tumour treatment.


Serotoninergic stimulation of aldosterone secretion in vivo: role of the hypothalamo-pituitary adrenal axis. The control of aldosterone secretion in vivo by serotonin was studied in conscious rats. Serial blood samples were taken from indwelling arterial cannulae before and after i.p. administration of 1 ml (4 g/l) 5-hydroxytryptophan (5-HTP), the precursor of serotonin (5-HT), or saline, and analysed for 5-HTP, serotonin, 5-hydroxyindoleacetic acid, plasma renin activity (PRA), corticosterone, aldosterone, sodium and potassium concentration. The relative contribution of the hypothalamo-pituitary adrenal axis was investigated in animals pretreated with the synthetic glucocorticoid dexamethasone. 5-HTP caused a significant increase in all parameters within 45 min except for plasma sodium and potassium. Saline administration showed no significant effect. Dexamethasone pretreatment significantly impaired the corticosterone and aldosterone response to 5-HTP, although the aldosterone response was merely attenuated. No other parameter was affected by dexamethasone pretreatment. The results show that administration of 5-HTP, which increases serum serotonin levels, stimulates PRA, corticosterone and aldosterone secretion. Dexamethasone pretreatment inhibits the aldosterone response, though not completely, suggesting that the stimulatory action of 5-HTP involves the release of ACTH, which stimulates corticosterone and aldosterone secretion by the adrenal cortex. The failure of dexamethasone to block the aldosterone response completely, suggests the involvement of other mechanisms such as the renin-angiotensin system or a direct action of serotonin on the adrenal zona glomerulosa.


Androgens and androgen-receptors in prostate tissue from patients with benign prostatic hyperplasia: effects of cyproterone acetate. Testosterone, 5 alpha-dihydrotestosterone and cyproterone acetate (CPA) were estimated in samples of prostate tissue, obtained from benign prostatic hyperplasia (BPH) patients who were or were not pretreated with CPA. Furthermore, these steroids were estimated in various fractions of the BPH tissue, and the number of nuclear androgen-receptor sites was determined. CPA-treatment caused a 4-fold, significant suppression of 5 alpha-dihydrotestosterone levels in total prostate tissue and its subfractions, without affecting testosterone levels or the androgen-receptor contents of the nuclear extracts. Nuclear concentrations of CPA were twice as high as those of 5 alpha-dihydrotestosterone. It is concluded that effects of CPA may have been caused through a combination of the following mechanisms: (1) suppression of peripheral androgen levels; (2) competition with androgens for (nuclear) androgen-receptors; and (3) suppression of prostatic 5 alpha-reductase.


Involvement of the macrophage low density lipoprotein receptor-binding domains in the uptake of oxidized low density lipoprotein. Macrophages, unlike most other cells, possess both low density lipoprotein (LDL) and scavenger receptors. The scavenger receptor has been shown to mediate the uptake of oxidized LDL (ox-LDL), which ultimately leads to cholesterol loading of the macrophages. The present study was undertaken to define epitopes on ox-LDL that are important for lipoprotein binding to macrophages and to ascertain whether ox-LDL can bind to the LDL receptor. Monoclonal antibodies (Mabs) directed against several epitopes along the apolipoprotein B-100 (apo B-100) molecule were used. LDL (300 micrograms/ml) was oxidized by incubation with 10 microM CuSO4 for 24 hours. Ox-LDL, as opposed to acetylated LDL (ac-LDL), reacted with Mabs directed against the LDL receptor-binding domains (Mabs B1B6 and B1B3). Similarly, uptake of ox-LDL but not ac-LDL by a murine J774 macrophage-like cell line was inhibited by as much as 40% after using Mab B1B6. The anti-LDL receptor antibody IgG-C7 also inhibited 125I-ox-LDL uptake by macrophages by 60%. Chromatography on heparin-Sepharose columns of LDL that was partially oxidized for only 3 hours resulted in two fractions: an unbound fraction with characteristics similar to those of ox-LDL and a bound fraction similar to native LDL. Macrophage degradation of the unbound fraction was inhibited by Mab IgG-C7 and Mab B1B6, which are directed toward the LDL receptor and the LDL receptor-binding domains on apo B-100, respectively. When incubated with three types of macrophages, J774 macrophage cells, mouse peritoneal macrophages, and human monocyte-derived macrophages, excess amounts of unlabeled ox-LDL, like native LDL but unlike ac-LDL, substantially suppressed the uptake and degradation of 125I-labeled LDL. Similar studies with fibroblasts, however, revealed that unlabeled LDL but not unlabeled ox-LDL or ac-LDL competed with 125I-LDL for cellular uptake and degradation. Mab directed against epitopes on the amino terminus domain of apo B-100 (C14) demonstrates a similar immunoreactivity with ox-LDL and native LDL but a much lower reactivity with ac-LDL. Mab C14 inhibited macrophage degradation of ox-LDL by 34% but had no inhibitory effect on the uptake of native LDL or ac-LDL. Thus, the ac-LDL and LDL receptor-binding domains as well as a unique epitope on the amino terminus of apo B-100 may be involved in macrophage binding of ox-LDL.(ABSTRACT TRUNCATED AT 400 WORDS)


Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies. This study represents a systematic analysis of the distribution of collagen types in human atherosclerotic lesions. Formalin-fixed, paraffin-embedded aortic tissues of 40 lesions from 16 different individuals ranging in age from 1 month to 84 years were examined immunohistochemically using antibodies to type I, III, IV, V, and VI collagens. Preembedding immunoelectron microscopy was used to simultaneously localize type V and VI collagens within the lesions. Localization of type III collagen was very similar to that of type I, and type VI collagen appeared together with these two types of collagen in the thickened intimas of all stages of the lesion. Type V collagen was not detected in either fatty streaks or the mild intimal thickening of the aortas of children. With advancing age and lesion progression, the immunoreactivity with anti-type V collagen antibody became more intense. Type IV collagen was detected in the basement membrane region of intimal cells. In advanced lesions thick deposits of type IV collagen were found around the elongated smooth muscle cells. Using immunoelectron microscopy, type V collagen was found to be localized to cross-banded collagen fibers, and type VI collagen was found to be localized to beaded filaments present throughout the interstitium of the thickened intima. These findings suggest that collagens preserve the pathophysiological and functional integrity of the vascular wall by providing mechanical support as well as assuring the proper interaction of cells during the formation of atherosclerotic lesions.


Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake. A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.


Oncogene RNA expression in three human lymphoblastic leukemia cell lines lymphocytes. We have explored the RNA oncogene expression by three human acute lymphoblastic leukemia lines, NALM-6, MOLT-3, and REH. We compared such expression with that of normal human lymphocytes. Marked differences in the RNA expression of the third exon of c-myc, v-myb, v-Hras, N-ras, v-fes, and v-fos were found. We noted minimal or no differences in the RNA expression of v-abl, B-lym, v-erb-B, c-ets, v-fms, v-kras, v-mos, v-raf, and v-sis.


A human skeletal muscle cell line obtained from an adult donor. A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated markers. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for alpha-bungarotoxin (Bmax = 0.7 pmol/mg protein, Kd = 1.5 nM) and dihydropyridines (Bmax = 0.3 pmol/mg protein, Kd = 0.5 nM for [3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.


Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation. Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.


Topographic relationship between senile plaques and cerebrovascular amyloidosis in the brain of aged dogs. The distributions of senile plaques (SP) and cerebrovascular amyloidosis (CA) were studied by employing thioflavin S and modified Bielschowsky stains, and beta-protein immunohistochemistry on serial sections of the brains of aged dogs older than 10 years. Mature and perivascular plaques, both of which contained compact amyloid deposits, always showed a close topographic relationship to CA. In contrast, the majority of diffuse plaques showed no topographic relationship to CA. Cell bodies of neurons and/or glia were almost always involved in the diffuse plaques. In addition, beta-protein immunohistochemistry demonstrated amyloid deposits on the periphery of occasional neurons. These findings suggest that different mechanisms may be involved in the development of the different subtypes of SP in the brains of aged dogs.


Differential gene expression during germination and after the induction of adventitious bud formation in Norway spruce embryos. A pulse treatment of embryos of Norway spruce with cytokinin suppresses germinative development and induces the coordinate formation of adventitious buds from subepidermal cell layers. To analyse the patterns of gene expression associated with germination and the alterations induced by the bud induction treatment, we have isolated cDNA clones corresponding to genes that are differentially expressed in cytokinin-treated and untreated in vitro germinating embryos. One category of 14 clones hybridized to transcripts that were abundant specifically during germination. The expression of 8 of these genes was reduced by the bud induction treatment. Four clones, including one identified as a histone H2A gene, recognized transcripts that showed an increased abundance in bud-induced versus in vitro germinating embryos. A second category of 13 clones hybridized to transcripts that increased in abundance during post-germinative development of the seedling. Among these a subset of 8 clones, including an alpha-tubulin clone, corresponds to genes suppressed by the bud induction treatment, whereas 5 clones, including a gene with sequence similarity to polyubiquitin, were unaffected by the treatment. One clone hybridized to a message abundant in the seed, during early germination as well as in the vegetative bud, and showed 60% partial sequence identity to a barley (1----3)-beta-glucanase gene. Genes expressed exclusively in bud-induced or in vitro germinating embryos were not found. The results show that a major difference in gene expression between treated and untreated embryos is related to the shift from extensive cell proliferation to elongation and differentiation that occurs at the transition from germination to post-germinative development, and which is suppressed in the bud-induced embryos.


Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts. Expression cassettes containing a duplicated cauliflower mosaic virus (CaMV) 35S promoter fused to a polylinker preceded by the CCACCATGG and AACAATGG sequences were constructed. These two sequences correspond to the consensus sequences around the translation start codons in vertebrates and plants respectively. Translational fusions were made with the beta-glucuronidase-coding sequence and transient expression was recorded in tobacco mesophyll protoplasts. Approximately three times more GUS activity was found in protoplasts incubated with the constructs harbouring translational fusions as compared to a control harbouring a transcriptional fusion. No significant difference was observed between GUS activities obtained with the two consensus sequences.


Dynamics and localization of early B-lymphocyte precursor cells (pro-B cells) in the bone marrow of scid mice. Mice homozygous for the scid (severe combined immunodeficiency) mutation are generally unable to produce B lymphocytes, a condition attributed to defective rearrangement of immunoglobulin genes in precursor B cells. Some early B-lineage cells are present in the bone marrow (BM), however. In scid mice, we defined three subsets of early progenitor B cells lacking mu heavy chains (pro-B cells) based on the expression of terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein: (a) early pro-B cells (TdT+B220-), (b) intermediate pro-B cells (TdT+B220+), and (c) late pro-B cells (TdT-B220+). Double immunofluorescence labeling of BM cell suspensions has shown normal numbers of early and intermediate pro-B cells, substantially reduced numbers of late pro-B cells, and an absence of pre-B cells and B cells. Early and intermediate pro-B cells accumulated in metaphase in near-normal numbers after intraperitoneal (IP) vincristine administration. B220+ pro-B cells have been localized in BM sections by the binding of intravenously (IV) administered 125I monoclonal antibody (MoAb) 14.8, detected by light and electron microscope radioautography. Many B220+ cells were located peripherally in the bone-lining cell layers associated with stromal reticular cells. More centrally located B220+ cells were frequently associated with macrophages containing prominent cytoplasmic inclusions. Occasional B220+ cells were present in venous sinusoids. These results demonstrate that many pro-B cells in scid mice occupy microenvironments in the BM near the surrounding bone. The pro-B cells maintain normal rates of production during stages of presumptive mu heavy-chain gene rearrangement, apparently unaffected by the absence of a mature B cell pool. Nearly all defective cells then abort at the late pro-B cell stage and are deleted, apparently by macrophages. The findings contribute to models of in vivo differentiation, regulation, localization, and selection of early B-lineage cells in the BM.


A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth rat. Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.


CD4+CD7-CD57+ T cells: a new T-lymphocyte subset expanded during human immunodeficiency virus infection. CD7 and CD57 are two cell surface molecules related to the differentiation or functional stages of CD4+ T cells. The CD4+CD7- T cells represent a minor subset of CD4+ cells in normal individuals and are considered to contain the normal counterpart of Sézary T cells; the CD4+CD57+ peripheral blood lymphocytes (PBL) are detectable in long-term renal allograft recipients. We compared the cell surface expression of these CD7 and CD57 markers on CD4+ T lymphocytes in peripheral blood and lymphoid organs from normal individuals and human immunodeficiency virus (HIV)-infected patients. Our results indicate that CD4+CD7- T cells in normal PBL do not express CD57 and were poorly responsive to anti-CD3 monoclonal antibody (MoAb), the activation being restored by addition of anti-CD28 MoAb. This CD4+CD7- cell subset is increased in peripheral blood during HIV infection, and its progressive expansion mirrors both the absolute and relative decrease of CD4+ T cells. The lack of CD7 expression is correlated with CD57 acquisition on CD4+ T cells because CD4+CD7-CD57+ cells represent a major component of the CD4+CD7- subset in HIV-infected patients. Our results suggest that the presence and the expansion of CD4+CD7-CD57+ T lymphocytes, which do not behave as previously defined helper subsets, may participate to the immune dysfunction observed during HIV infection.


Acquired immunodeficiency syndrome-associated T-cell lymphoma: evidence for human immunodeficiency virus type 1-associated T-cell transformation. The majority of lymphomas in the setting of acquired, iatrogenic, or congenital immunodeficiencies are B-cell lymphoproliferations. We describe a rare T-cell lymphoma in a fulminantly ill patient infected with human immunodeficiency virus type 1 (HIV-1). The T-cell nature of the process was defined genotypically (monoclonal T-cell receptor beta-chain [CT beta] rearrangement) and phenotypically (CD45RO+, CD4+, CD5+, CD25+, CD8-, CD3- and negative for a variety of B-cell and monocyte markers). The CD4+, CD25+ (interleukin-2 receptor [IL-2R]) phenotype with production of IL-2 and IL-2R RNA is analogous to human T-lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATLL); however, no HTLV-1 could be detected. Southern blot analysis did demonstrate monoclonally integrated HIV-1 within the tumor genome. Furthermore, the tumor cells were producing HIV p24 antigen as shown by immunohistochemistry. This is the first case of acquired immunodeficiency syndrome (AIDS)-associated non-Hodgkin's lymphoma in which HIV-1 infection may have played a central role in the lymphocyte transformation process.


Detection and localization of Epstein-Barr viral genomes in angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma. We studied 23 cases of angioimmunoblastic lymphadenopathy (AILD) and AILD-like lymphoma for evidence of Epstein-Barr virus (EBV) using the polymerase chain reaction (PCR) and in situ hybridization studies. EBV nucleic acid sequences were found by either PCR or in situ hybridization in 96% of the cases. There was a wide range in the number of EBV-positive cells among the different cases as detected by in situ hybridization. The EBV-positive cells most often possessed nuclei of intermediate to large size. Double-labeling immunohistochemistry/in situ hybridization studies demonstrated that most of the EBV-positive cells expressed the B-lineage antigen CD20 (as detected by L26), with a minority of the EBV-positive cells stained for the T-lineage associated antigen, CD43 (as detected by Leu 22). The abnormally high amounts of EBV found in AILD and AILD-like lymphoma may be a reflection of decreased immunocompetence in these patients. The presence of EBV-positive B cells may explain the presence of B-cell clones found by others as well as the paradoxical occurrence of B-cell lymphoma in a primary T-cell lymphoproliferative disorder.


Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia. Acute myelogenous leukemia (AML) is a clonal disease that is heterogeneous with respect to the pattern of differentiative expression of the leukemic progenitors. In some patients, the involved stem cells manifest pluripotent differentiative expression, whereas in others, the involved progenitors manifest differentiative expression mainly restricted to the granulocytic pathway. This is in contrast to chronic myelogenous leukemia (CML) which is a clonal disease known to arise in a pluripotent stem cell. Therefore, we tested whether these leukemias could be distinguished with respect to their involvement of immature precursors by studying colony-forming cells (CFC) and their precursors from four glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML and five patients with CML. CFC were separated from their precursors by FACS for expression of CD33 and CD34 followed by growth in a long-term culture (LTC) system. The vast majority of CFC express both the CD33 and CD34 antigens, but their less mature precursors, detected by their ability to give rise to CFC in LTC, express only CD34. In three of the four patients with AML, the CD33-CD34+ cells produced CFC in LTC that appeared to be predominantly or completely normal (ie, nonclonal) in origin. In the fourth patient, a significant enrichment of nonclonal progenitors was obtained in the CD33-CD34+ population, but these cells may also have included significant numbers of clonal cells. In contrast, in four of five patients with CML, cultures of both the CD33-CD34+ and CD33+CD34+ populations produced CFC in LTC that were almost entirely clonal in origin, whereas in the fifth patient a substantial number originated from nonclonal stem cells. These data indicate that granulocyte/monocyte progenitors are predominantly clonally derived in CML and AML. In CML, their precursors are also predominantly clonal, but in some cases of AML they are not. These findings may have implications for understanding the success or failure of current therapies of AML and CML.


Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry. Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF-negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte-poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.


Influence of steel factor on hemoglobin synthesis in sickle cell disease. A new hematopoietic growth factor (Steel factor) has been identified which stimulates erythroid proliferation both in vitro and in vivo. We evaluated the influence of recombinant Steel factor on hemoglobin synthesis in peripheral blood (PB) BFU-E-derived cells from normal donors by radioimmunoassay (RIA) and compared it with stimulation with GM-CSF and interleukin-3 (IL-3). Only Steel factor stimulated a significant increase in BFU-E-derived colony size and a significant increase in fetal hemoglobin (HbF) in BFU-E-derived erythroblasts from 0.49% +/- 0.27% to 6.33% +/- 1.11% in serum-deprived media and from 1.88% +/- 0.24% to 11.17% +/- 0.91% in serum. To determine whether this influence on hemoglobinization also occurred in sickle cell disease, we studied 13 patients with sickle cell disease. In serum-deprived conditions, there was a significant increase in the number and size of BFU-E-derived colonies with Steel factor that was dose-dependent. In addition, the proportion of HbF in progenitor-derived cells increased by 66% from 4.1% +/- 0.6% to 6.8% +/- 1.2% with Steel factor. In serum-containing conditions studied in 12 patients, the increase in percentage of HbF was even greater, from 10.7% +/- 0.9% in control cultures to 22.5% +/- 2.6% with Steel factor. These increases in percentage of HbF were significant and dose-dependent. An increase in percentage of HbF was observed in erythroblasts harvested on day 11, 14, and 18 of culture. A decrease in mean picograms of total Hb per cell after coculture with Steel factor was noted, suggesting that growth kinetics influenced complete hemoglobinization. In serum-deprived conditions, picograms of HbF per cell was not affected by Steel factor, and in serum-containing conditions that augment in vitro HbF production it was enhanced. Thus, Steel factor stimulated a significant increase in percentage of HbF in erythroid cells from normal donors and patients with SCA in vitro.


Circulating granulocyte colony-stimulating factor (G-CSF) levels after allogeneic and autologous bone marrow transplantation: endogenous G-CSF production correlates with myeloid engraftment. Myeloid engraftment after bone marrow transplantation (BMT) is influenced by a number of variables, including cytoreductive chemoradiotherapy, genetic disparity, number of reinfused committed myeloid progenitor cells, healthy microenvironment, and the presence of hematopoietic growth factors. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation of myeloid progenitor cells and enhances myeloid engraftment after BMT. We investigated the temporal relationship between endogenous G-CSF production and myeloid engraftment in both children and adults after allogeneic (ALLO) and autologous (AUTO) BMT. Circulating endogenous G-CSF levels ranged between 0 and 2552 pg/mL. The correlation coefficient between circulating serum G-CSF levels and the peripheral absolute neutrophil count (ANC) was r = -.875 (P less than .001). The endogenous serum G-CSF level was highest during the first week after BMT, when the ANC was less than or equal to 200/microL (699 +/- 82.3 pg/mL) (P less than .001). Both children and adults demonstrated a similar inverse relationship between circulating G-CSF level and degree of neutropenia. One patient failed to engraft after AUTO BMT and also failed to generate any endogenous G-CSF production. Lastly, once the serum G-CSF level decreased to less than 200 pg/mL, a mean of 6.1 +/- 0.9 days elapsed before the ANC was greater than or equal to 500/microL for 2 consecutive days. This study demonstrates that endogenous G-CSF production is associated with myeloid engraftment in both children and adults after AUTO and ALLO BMT and that the rate of increase and decrease in endogenous G-CSF may be predictive of either failure to engraft or duration of neutropenia.


Characterization of Shiga-like toxin producing Escherichia coli (SLTEC) isolated from calves with and without diarrhoea. To determine if shiga-like toxin producing Escherichia coli (SLTEC) are involved in neonatal calf diarrhoea, isolated E. coli strains from diarrhoeic and non-diarrhoeic calves were characterized for shiga-like toxin (SLT) by colony blot hybridization and cytotoxicity assays. None of 150 E. coli strains isolated from diarrhoeic calves in 1985-1988 was positive for SLT, while 7/232 (3.0%) isolated in 1989 were positive for SLT. In contrast, samples collected during 1989 and 1990 from diarrhoeic calves were 21.9% SLTEC positive, and samples from non-diarrhoeic calves were 12.9% SLTEC positive. SLT I positive E. coli strains were isolated more often from diseased (17.8%) than from healthy animals (5.0%), while SLT II positive E. coli were more often detected in non-diarrhoeic (8.9%) than in diarrhoeic calves (4.1%). The mean percentage of SLT I positive E. coli in the whole E. coli flora of the samples was significantly higher in diarrhoeic than in healthy animals, implying a pathogenic role of SLT I producing E. coli in neonatal calf diarrhoea. Enterohemolysin was produced by 70.8% of the SLT I producing E. coli strains examined. Determination of O- and K-antigens of SLT positive E. coli revealed a highly diverse spectrum of SLTEC O-groups in calves. While no E. coli isolate belonged to serotype O157:H7, classical human enteropathogenic E. coli O-groups (O26, O111, O128) were detected. These results support the theory that cattle serve as a reservoir for human SLTEC infection.


Biochemical and serological patterns of Escherichia coli O2 strains isolated from patients with urinary tract infections. We examined the results of two biochemical test systems for their ability to discriminate a series of 58 Escherichia coli O2 strains collected from patients with urinary tract infections. The O:K:H serotypes and O antigen factors of the strains were also determined. The strains could be assigned to 13 distinct serological patterns by means of O2 antigen factors as well as K and H antigens. The combination of O:K:H-serotyping with biotyping enabled very fine strain discrimination. These data indicate that the biotyping system of Achtman (BTA) is more precise than the Enterobacteriaceae-code-system described by Reiske (ECR) and that the determination of BTA types supports the identification of bacterial clones.


Inhibition of neurogenic plasma exudation in guinea-pig airways by CP-96,345, a new non-peptide NK1 receptor antagonist. A new non-peptide tachykinin antagonist, CP-96,345, inhibited airway plasma exudation induced in guinea-pigs by i.v. substance P in a dose-dependent manner with dose-ratios in the main bronchi of 5 at 1 nmol kg-1 and 19 at 100 nmol kg-1. At 100 nmol kg-1, CP-96,345 completely inhibited plasma exudation induced by either electrical stimulation of the cervical vagus nerves or i.v. capsaicin, indicating inhibition of the effects of endogenous tachykinins, but did not inhibit the bronchoconstrictor response to neurokinin A, suggesting selectivity for NK1 receptors. CP-96,345 may be useful in examining the role of endogenous tachykinins in vivo.


The effects of chronic treatment with the dihydropyridine, Bay K 8644, on hyperexcitability due to ethanol withdrawal, in vivo and in vitro. 1. The effects of chronic treatment with the dihydropyridine, Bay K 8644, were studied on the ethanol withdrawal syndrome, in vivo and in vitro. 2. Addition of racemic Bay K 8644 to the drinking mixture, throughout the chronic ethanol treatment, decreased the behavioural excitability seen during ethanol withdrawal in vivo. 3. All the signs of hyperexcitability in field potentials in the isolated hippocampal slice, caused by ethanol withdrawal, were decreased by the chronic administration of Bay K 8644. 4. These effects resembled those previously reported for chronic administration of calcium channel antagonists; racemic Bay K 8644 has both calcium channel activating and antagonist properties. 5. Measurement of brain levels of Bay K 8644 at the end of the chronic treatment showed that the compound reached micromolar concentrations during the treatment, but none could be detected in the tissues at the time of the above measurements. 6. It is possible that the results might be explained by predominance of the calcium channel antagonist properties of this compound, owing to the high central concentrations achieved during the treatment. Tolerance to the calcium channel activating properties of Bay K 8644 may also have occurred during the chronic treatment.


Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava. 1. Whole-cell patch-clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2. Depolarizing pulses, applied from a holding potential of -90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mM Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of -80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nM) while racemic isradipine inhibited the current (IC50 = 21 nM). 3. The voltage-dependency of isradipine blockade was assessed by determining the steady-state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nM for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)-[3H]-isradipine obtained in intact strips incubated in 5.6 mM or 135 mM K+ solutions confirmed the voltage-dependency of isradipine binding. 4. Specific binding of (+)-[3H]-isradipine was completely displaced by unlabelled (+/-)-isradipine, with an IC50 of 15.1 nM. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nM) in cells maintained at a holding potential of -80 mV. 5. Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady-state inactivation-activation curves of Ba2+ current were not significantly shifted along the voltage axis.6. The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.


M-currents in frog sympathetic ganglion cells: manipulation of membrane phosphorylation. 1. The inward current and the M-current (IM) suppression produced when muscarine is applied to frog sympathetic ganglion cells was recorded by means of the whole-cell patch-clamp technique. The holding potential was -30 mV and [K+]o was 6 mM. 2. The steady-state IM was maintained for at least 20 min when the patch pipette contained neither adenosine 5'-triphosphate (ATP) nor adenosine 3':5'-cyclic monophosphate (cyclic AMP). Inclusion of these substances or the ATP antagonist, beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-MethATP; 1 or 2 nM) (failed to alter the rate of IM 'run down'. By contrast, inclusion of adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S, 1 or 2 mM) resulted in a 60% reduction of the current within 18 min. 3. Despite the inability of ATP-gamma-S to maintain steady-state IM, it had no effect on the ability of muscarine (2-100 microM) to suppress a constant fraction of the available current. ATP-gamma-S and beta,gamma-MethATP increased the rise time and duration of the response to muscarine. 4. Inclusion of a phosphatase inhibitor, diphosphoglyceric acid (DPG, 1-2.5 mM) or alkaline phosphatase (100 micrograms ml-1) failed to affect the amplitude of muscarinic responses. 5. These results question the role of the phosphorylation and/or dephosphorylation reactions in the transduction mechanism for muscarine-induced IM suppression but are consistent with the possibility that M-channels are 'directly coupled' via G-protein to the muscarinic receptor.


Effects of cromakalim on the contraction and the membrane potential of the circular smooth muscle of guinea-pig stomach. 1. The effects of cromakalim on mechanical and electrical activities of the circular smooth muscles of guinea-pig stomach antrum were observed. 2. Cromakalim (greater than 1 x 10(-7) M) decreased the amplitude of spontaneous rhythmic contractions and also the acetylcholine-enhanced spontaneous contractions. Cromakalim was less effective against the 25.9 mM and 35.9 mM K(+)-induced tonic contractions. 3. Glibenclamide (1 x 10(-6) M) itself caused no detectable change in the spontaneous contractions, those potentiated by acetylcholine or tonic contractions induced by high K+ solutions, but attenuated the actions of cromakalim. On the other hand, charybdotoxin (3 x 10(-8) M) increased the amplitude of spontaneous contractions but failed to affect the actions of cromakalim. 4. Cromakalim (greater than 1 x 10(-6) M) decreased the amplitude and duration of slow waves, and hyperpolarized the membrane. These actions of cromakalim were completely antagonized by 1 x 10(-6) M glibenclamide, whereas part of the effects of cromakalim on mechanical activity was resistant to glibenclamide. 5. The results suggest that the inhibition by cromakalim of the electrical activity and the hyperpolarization, which may be associated with the opening of glibenclamide-sensitive K+ channel, are responsible for its inhibitory action on circular smooth muscle of guinea-pig stomach. Further, some effects independent of glibenclamide-sensitive K+ channel may also be responsible for the mechanical effect.


Differential inhibitory effects of opioids on cigarette smoke, capsaicin and electrically-induced goblet cell secretion in guinea-pig trachea. 1. Goblet cell secretion in guinea-pig airways is under neural control. Opioids have previously been shown to inhibit neurogenic plasma exudation and bronchoconstriction in guinea-pig airways. We have now examined the effects of morphine and opioid peptides on tracheal goblet cell secretion induced by either electrical stimulation of the cervical vagus nerves, exogenous capsaicin, or acute inhalation of cigarette smoke. The degree of goblet cell secretion was determined by a morphometric method and expressed as a mucus score which is inversely related to mucus discharge. 2. Morphine, 1 mg kg-1, completely blocked goblet cell secretion induced by electrical stimulation of the vagus nerves. Morphine also inhibited the response to cigarette smoke given either at a low dose (10 breaths of 1:10 diluted in air), which principally activates cholinergic nerves, or at a high dose (20 breaths of undiluted), which activates capsaicin-sensitive sensory nerves, by 100% and 73% respectively. In contrast, morphine had no significant inhibitory effect on capsaicin-induced goblet cell secretion. The inhibitory effect of morphine was reversed by naloxone. 3. Selective mu- or delta-opioid receptor agonists, [D-Ala2, NMePhe4, Glyol5]enkephalin (DAMGO) or [D-Pen2, D-Pen5]enkephalin (DPDPE) respectively, caused a dose-related inhibition of low dose cigarette smoke-induced goblet cell discharge, with DPDPE more potent than DAMGO. A kappa-receptor agonist, trans-3,4-dichloro-N-methyl-N-(2-(1-pyrollidinyl)cyclohexyl) benzeneacetamine (U-50,488H), had no inhibitory effect. DPDPE had no inhibitory effect on goblet cell secretion induced by exogenous methacholine. 4. DAMGO dose-dependently blocked the response to high dose cigarette smoke with a maximal inhibition of 95% at 2 x 10(-7) mol kg-1. Neither DPDPE nor U-50,488H had any significant inhibitory effect. The increase in goblet cell secretion induced by exogenous substance P was not affected by DAMGO.5. We conclude that opioids inhibit neurally-mediated goblet cell secretion via actions at prejunctional delta and mu-receptors on cholinergic nerves and at mu-receptors on sensory nerve endings, and that capsaicin activation of sensory nerves is via a different mechanism from that of electrical or cigarette smoke activation.


An ubiquitous modulating function of rabbit tracheal epithelium: degradation of tachykinins. 1. To examine the role of epithelium in the responsiveness of tracheal smooth muscle in rabbit, we measured the contractile responses to acetylcholine (ACh), KCl, 5-hydroxytryptamine (5-HT), histamine, substance P (SP), neurokinin A (NKA), and electrical field stimulation  EFS) in intact and epithelium-denuded preparations. 2. Removal of epithelium did not alter the contractile response to any agonist examined, except SP. 3. Removal of epithelium enhances the contractile response to SP. In the presence of phosphoramidon, the contractile response to SP was not significantly different in either group. The results suggest that the effect of epithelium is largely due to degradation of SP by enzymes in the epithelium. 4. Arachidonic acid metabolites did not seem to be related to the responses induced by contractile agonists or EFS. 5. In the presence of SP, the contractile responses and [3H]-choline outflow evoked by EFS were dose-dependently enhanced. Contractile responses to EFS and [3H]-choline outflow evoked by EFS were enhanced by SP significantly more than by NKA. Both were abolished by atropine or tetrodotoxin. 6. These results suggest that rabbit tracheal epithelium may modulate SP-induced contractions, both direct and indirect, by inactivation of SP. This phenomenon is widespread in mammals. The rabbit may be a useful model to examine airway cholinergic functions.


Cytoprotection by iloprost against paracetamol-induced toxicity in hamster isolated hepatocytes. 1 The ability of iloprost (ZK36374) to protect hamster isolated hepatocytes from the toxic effects of paracetamol and its reactive metabolite N-acetyl-p-benzoquinoneimine (NABQI) was investigated. The cytoprotection provided by iloprost was compared with that of N-acetyl-L-cysteine. 2 Treatment of hepatocytes with either NABQI (0.4 mM) or paracetamol (2 mM) alone resulted in a considerable loss of cell viability, as assessed by trypan blue exclusion or leakage of lactate dehydrogenase, accompanied by an increase in the percentage of viable cells that were blebbed. N-acetyl-L-cysteine (1.25 mM) pretreatment diminished the loss of cell viability and the percentage of blebbed cells resulting from exposure to NABQI or paracetamol, whereas iloprost (10(-16) M to 10(-10) M) pretreatment reduced only the loss of cell viability, not the percentage of viable cells exhibiting blebbing. Pretreatment with N-acetyl-L-cysteine significantly attenuated the depletion by paracetamol of glutathione and decreased the covalent binding of [14C]-paracetamol to cellular proteins, whereas iloprost was without any such effects. 3 The effects of iloprost and N-acetyl-L-cysteine were also investigated by use of a model of paracetamol toxicity in which it is possible to study the biochemical events leading to cell injury separate from the generation of toxic metabolites. Hamster hepatocytes were incubated with paracetamol (4 mM) for 90 min at 37 degrees C during which metabolism of paracetamol occurs with minimal loss of cell viability. Following washing of cells, to remove paracetamol and its metabolites, there was a progressive loss of viability and increase in the percentage of cells exhibiting blebbing when incubated in buffer alone. Addition of either N-acetyl-L-cysteine (1.25 mM) or iloprost (10 14M to 10 -M), following washing, significantly reduced the expected loss of cell viability. Iloprost at concentrations outside this range was without effect.4. Paracetamol toxicity to isolated hepatocytes could be prevented or delayed by treatment with either N-acetyl-L-cysteine or iloprost, but whereas the former prevented or even reversed plasma membrane blebbing with a resultant reduction in the percentage of viable cells that were blebbed, the prostanoid appeared only to delay the progression from plasma membrane blebbing to loss of viability. Hence, the percentage of viable cells that were ultimately blebbed following exposure to paracetamol was not significantly reduced by addition of iloprost.5. Aspirin or ibuprofen exacerbated the loss of viability induced by prior incubation with paracetamol. Thus, there may be a role for endogenous prostaglandins in protecting hepatocytes from paracetamol toxicity.6. Iloprost is cytoprotective without any effect upon toxin metabolism or detoxication. The mechanism of action of iloprost probably does not involve induction of prostaglandin synthesis or activation of the previously-characterized prostacyclin receptor.


Different patterns of release of endothelium-derived relaxing factor and prostacyclin. 1. Release of endothelium derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells (EC) cultured from bovine aortae was measured by bioassay and radioimmunoassay, respectively, during infusions (10 min) of bradykinin (BK), adenosine diphosphate (ADP), arachidonic acid (AA), alkaline buffers and the free-bases (FB) of L-arginine or D-arginine. Release of EDRF from the luminally perfused rabbit aorta was also measured during infusions (10 min) of acetylcholine (ACh), substance P and ADP. 2. Bradykinin (10 or 30 nM) infused through the column of EC induced release of both EDRF and PGI2, neither of which was maintained for the duration of the infusion. 3. ADP (1.6 or 4 microM) infused through the column of EC induced release of a EDRF which was maintained for the duration of the infusion and a release of PGI2 which lasted for a much shorter period. 4. Arachidonic acid (30 or 90 microM) infused through the column of EC caused a sustained release of EDRF and PGI2, both of which outlasted the infusion of AA. 5. L-Arginine FB, D-arginine FB or alkaline buffer infused through the column of EC released EDRF, but only small amounts of PGI2. The release of EDRF outlasted the period of infusion and was due to an increase in the pH of the Krebs solution perfusing the EC. 6. Infusions of ACh (0.25-1 microM) or ADP (4-16 microM) caused a sustained release of EDRF from the luminally-perfused rabbit aorta, whereas infusion of substance P (3.3-10 microM) caused only a transient release of EDRF. 7. These results show that distinct patterns of EDRF release exist to different agonists in both cultured and in situ EC, and that EDRF and PGI2 do not necessarily follow the same time course of release. Furthermore, sustained release of EDRF does not require the constant infusion of the precursor, L-arginine, whereas sustained release of PGI2 only occurs when AA, the precursor of PGI2, is present in the extracellular medium.


Mechanical restitution and post extrasystolic potentiation of perfused rat heart: quantitative comparison of normal right and left ventricular responses. Interval-force relationship of right and left ventricles of the isolated perfused rat heart was quantified by fitting polynomial, linear and mixed linear-exponential functions to the mechanical restitution (MRC) and post extrasystolic potentiation (PESPC) curves. Ventricular maximum developed pressure (Pmax) and its first derivative (dP/dtmax) were used as indices of contractility. MRCs and PESPCs could be separated into two distinct phases: phase A and phase B of MRCs; phase I and phase II of PESPCs. These phases for the right and left ventricle of the rat heart could be explained on the same model of cellular kinetics of the activator calcium, but showed distinct differences from other species. Right and left ventricle inotropic reserve (CRmax), as quantified from the centre of mass of the phase B of MRCs (from normalized Pmax), was (mean +/- SE): 132.4 +/- 2.05% and 132.1 +/- 1.7% at 1 Hz, which increased significantly (P less than 0.001) to 181.0 +/- 5.8% and 182.3 +/- 5.2% at 3.3 Hz, respectively. Linear regression of normalized right ventricle extrasystolic responses on the left ventricle responses gave a high correlation coefficient (typically r2 = 0.97). Time constants of the fitted mechanical restitution (TMRC) and post extrasystolic potentiation (TPESPC) curves were at 1 Hz, TMRC and TPESPC (from normalized dP/dtmax) were (mean +/- SE): 161.6 +/- 10.8 and 159.0 +/- 13.2 ms for right ventricle, and 196.1 +/- 14.5 and 188.3 +/- 10.7 ms for left ventricle, respectively. The results of this study indicate that interval-force relationship of the rat heart, as exemplified by CRmax and time constants of the fitted curves, could provide a useful index for quantifying and comparing right and left ventricular functions.