Steroid-induced myopathy and its significance to respiratory disease: a known disease rediscovered.

Skeletal muscle myopathy is a well-known side-effect of systemically administered corticosteroids. In recent years renewed attention is being paid to the involvement of the respiratory muscles and its consequent significance in pulmonary patients. Two different clinical patterns of steroid-induced muscular changes are known. In acute myopathy and atrophy after short term treatment with high doses of steroids, generalized muscle atrophy and rhabdomyolysis occur, including the respiratory muscles. Chronic steroid myopathy, occurring after prolonged treatment with moderate doses, is characterized by the gradual onset of proximal limb muscle weakness and may be accompanied by reduced respiratory muscle force. Animal studies demonstrated diaphragmatic myopathy and atrophy similar to the alterations in peripheral skeletal muscles. Fluorinated steroids induced selective type IIb (fast-twitch glycolytic) fibre atrophy, resulting in changes in contractile properties of the diaphragm. Non-fluorinated steroids may also induce histological, biochemical and functional alterations in the diaphragm. Observations in patients with collagen vascular disorders and with asthma and chronic obstructive pulmonary disease (COPD) underline the potential hazards of treatment with corticosteroids to respiratory muscle structure and function.


Perceived effect on shortness of breath of an acute inhalation of saline or terbutaline: variability and sensitivity of a visual analogue scale in patients with asthma or COPD.

The purpose of the study was to validate a bipolar visual analogue scale (VAS) to assess the perceived effect on shortness of breath of an acute inhalation and to search for differences in perception between asthmatics and subjects with chronic obstructive pulmonary disease (COPD). Thirty two subjects with airway obstruction and a diagnosis of either asthma (n = 16) or COPD (n = 16) received three consecutive inhalations of isotonic saline, followed by two inhalations of 400 micrograms terbutaline. Saline was perceived by asthmatics as a slight improvement: VAS (median, 95% confidence interval) 9%, 0-18% of line length. COPD subjects could be separated into two subgroups: "high perceivers" (n = 8, VAS 43%, 33-53%) and "low perceivers" (n = 8, VAS 5%, 3-7%). The median intrasubject coefficient of variation of the three post-saline VAS ratings was 19.4% (asthma), 12.5% (COPD high perceivers), and 14.5% (COPD low perceivers). After terbutaline, asthmatics had, by selection, a larger increase in forced expiratory volume in one second (FEV1) than COPD subjects. However, for other indices (expiratory specific resistance, inspiratory vital capacity and maximal inspiratory flow at 50% forced vital capacity) the changes were smaller in COPD low perceivers than in both asthmatics and COPD high perceivers. The parallel improvement in VAS was 24%, 20-39% (asthma), 15%, 6-25% (COPD high perceivers) and 1%, -1-8% (COPD low perceivers). The most sensitive index was FEV1 in asthmatics, vital capacity in COPD subjects, VAS being among the most sensitive indices in the former, but among the least sensitive in the latter. We conclude that the sensitivity of this VAS to bronchodilation is better in asthmatics than in COPD subjects. The latter can however be separated into subgroups with high and low level of perception.


Sequential treatment with low dose almitrine bismesylate in hypoxaemic chronic obstructive airways disease.

Daily dose schedules of 100-200 mg of almitrine bismesylate improve arterial blood gases in patients with hypoxaemic chronic obstructive airways disease (COPD) but dose related side effects are evident. In the present study, daily doses approximately half of those previously used were employed in a randomised double blind manner in 85 patients (age 35-79 years) with hypoxaemic COPD. After a one month period to check stability of arterial blood gases, patients were allocated to almitrine (A) or placebo (P) using an unequal code (60% A, 40% P). Tablets, 50-100 mg daily were stopped for one month after 3, 6 and 9 months to counteract drug accumulation. 50 patients in group A and 35 in group P were comparable on entry; mean age 65 (SD = 8) yrs., Pao2 7.8 (0.7) kPa (58.3 (5.0) mmHg), PaCO2 5.8 (0.8) kPa (43.2 (6.0) mmHg), forced expiratory volume in one second--FEV1 0.89 (0.25) l and 6 minute walking distance 296 (97) metres. The improvement in baseline PaO2 values was the same 0.8-1.3 kPa (6-9.8 mmHg) as with previous higher dose therapy. Approximately one third of patients did not respond, defined as PaO2 elevation > 0.67 kPa (5 mmHg). The sequential dosing scheme stabilised blood levels of almitrine within the therapeutic range of 280-300 ng.ml-1. After withdrawal of therapy arterial blood gases and spirometry reverted to pre-treatment levels, suggesting no permanent reversal of pathophysiology. Dose related side effects of breathlessness, indigestion and peripheral neuropathy were not observed. Nerve conduction studies revealed no difference in peripheral nerve dysfunction in hypoxaemic COPD between active and placebo therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


A twelve month comparison of salmeterol with salbutamol in asthmatic patients. European Study Group.

The efficacy and tolerability of salmeterol, 50 micrograms b.i.d. was compared for three months with salbutamol, 200 micrograms q.i.d., administered from metered-dose inhaler. For the following nine months, safety and clinic lung function was monitored on salmeterol, 50 micrograms b.i.d., compared with salbutamol, 200 micrograms b.i.d. This comparison was made in a multicentre, double-blind, parallel-group study of 667 moderate asthmatics, who had a forced expiratory volume in one second (FEV1) or peak expiratory flow rate (PEFR) > 50% predicted, a 15% reversibility to inhaled salbutamol and who were experiencing symptoms. Throughout the first three month treatment period, both morning and evening PEFR were significantly higher on treatment with salmeterol than salbutamol (mean differences between the treatments 30 l.min-1 for morning, p < 0.001, and 11 l.min-1 for evening, p < 0.01). In addition, the diurnal variation in PEFR, nocturnal and daytime symptoms and use of additional salbutamol were significantly lower in the salmeterol treated group. This improvement was also apparent in the separate subpopulations of patients taking no concurrent glucocorticosteroid or concurrent inhaled and/or oral glucocorticosteroids. Both treatments were well-tolerated throughout the 12 months of treatment. There was a lower incidence of asthma and related events during salmeterol treatment compared to salbutamol treatment subgroups. The results of the study clearly demonstrate that salmeterol, 50 micrograms b.i.d., is well-tolerated and more effective than salbutamol, 200 micrograms q.i.d., in the treatment of moderate asthma.


Easy bruising as a side-effect of inhaled corticosteroids.

We wished to determine the prevalence of easy bruising in patients taking inhaled corticosteroids (ICS) compared with those who do not. Differences in age, dosage and duration of use of ICS between patients who bruised and those who did not were also investigated. Confidential questionnaire surveys were conducted over a 6 month study period amongst patients attending a respiratory out-patient clinic and taking regular ICS, and a control group of patients attending non-respiratory clinics and not taking any form of corticosteroids. Patients with bleeding disorders or taking oral steroids, non-steroidal anti-inflammatory drugs or anticoagulants were excluded from the study. Questionnaires from 202 respiratory patients using ICS (group A) were compared with 204 non-ICS patients (Group B) of similar age and sex distribution. Significantly more patients in Group A reported easy bruising than in group B (47 vs 22%, relative risk 2.18, 95% confidence interval (95% CI) 1.62-2.94), and it was the commonest reported symptom. In Group A, the patients that reported easy bruising tended to be older (61 vs 52 yrs), on higher daily dosages (1,388 vs 1,067 micrograms) and had been taking inhaled corticosteroids for longer (55 vs 43 months) than non-bruisers. Overall, females reported easy bruising more frequently than males in both groups. However, comparing Group A with Group B, males taking ICS had a higher relative risk for bruising than females (males, relative risk 5.80, 95% CI 2.38-14.13; females, relative risk 1.80, 95% CI 1.32-2.44).(ABSTRACT TRUNCATED AT 250 WORDS)


Bronchial vagal tone and responsiveness to histamine, exercise and bronchodilators in adult patients with cystic fibrosis.

Atopy and bronchial responsiveness to histamine, exercise and bronchodilators were investigated in 18 adult patients with cystic fibrosis (CF). Reversibility of airflow limitation was measured after ipratropium bromide and terbutaline, and histamine and exercise provocation tests were performed. Histamine hyperresponsiveness was observed in 10 out of 18 patients and was not confined to those with severe airway obstruction. The positive histamine responders showed significantly better bronchodilatation after terbutaline, when compared to negative histamine responders. Histamine responsiveness was not related to atopy or exercise responsiveness. Exercise challenge caused bronchodilatation without bronchoconstriction in all patients. The exercise-induced bronchodilatation correlated with bronchodilatation after ipratropium bromide. It is proposed that an increased vagal tone may lead to an increased resting bronchomotor tone which can be reduced by ipratropium bromide and by exercise in adult patients with cystic fibrosis.


Comparison of nebulized and intravenous terbutaline during exacerbations of pulmonary infection in patients with cystic fibrosis.

Twenty three patients completed a double-blind study, comparing intravenous and nebulized terbutaline, during the first four days of a pulmonary exacerbation of cystic fibrosis (CF), with follow-up to day 10. Routine treatment with chest physiotherapy and appropriate intravenous antibiotics was given to all patients. The best peak flow rate (PF), forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), in the past year and at entry to the study, revealed no significant difference between the groups. However, on day 10, PF, FEV1 and FVC, of the nebulizer group remained significantly reduced compared to best values in the previous year, whereas the PF and FEV1 in the intravenous group were not significantly reduced compared to the best values in the previous year. Comparison of regression lines showing the overall rate of improvement of PF, FEV1 and FVC between the two groups showed that the rate of improvement of each parameter was more rapid in the group receiving intravenous terbutaline. This was statistically significant for PF. It is possible that during acute exacerbations of infection, sputum retention makes it more difficult for the inhaled bronchodilators to reach the airways and intravenous therapy is, therefore, more beneficial.


Effects of repeated exposure to 4 ppm nitrogen dioxide on bronchoalveolar lymphocyte subsets and macrophages in healthy men.

Following the basal descriptive studies of the bronchoalveolar inflammatory cell response induced by single exposure with nitrogen dioxide (NO2) in man, it was considered important to clarify the cell response to repeated exposure with NO2. This investigation was, therefore, undertaken with bronchoalveolar lavage (BAL) 3 weeks before and 24 h after six repeated exposures with 4 ppm NO2 (7 mg.m-3) in ten healthy volunteers. The exposures were performed during 20 min and every second day. Analysis of the recovered BAL fluid demonstrated that repeated exposures to NO2 caused a lung cell response different from that reported after a single exposure. Amounts of lavaged alveolar macrophages, B-cells, and natural killer (NK)-cells were decreased and the T-helper-inducer/cytotoxic-suppressor cell ratio was altered, but there was no lymphocytosis or mastocytosis as after single exposure. Lymphocyte numbers in peripheral blood were reduced after exposure. These results suggest that repeated exposure with NO2 adversely affects the immune defence. This could contribute to the increased susceptibility to airway infections reported to be associated with NO2 exposure.


Thymomodulin increases release of granulocyte-macrophage colony stimulating factor and of tumour necrosis factor in vitro.

To evaluate the effects of thymomodulin (TMD), a thymic biological response modifier derived from calf thymus, on the release of various cytokines involved in the lung immune reactions, human alveolar macrophages (AM) and peripheral blood lymphocytes (PBL) were cultured either alone or in co-cultures. In co-cultures of AM with PBL, TMD did not induce any change in gamma-interferon (gamma-IFN) and interleukin-1 (IL-1) secretion, while it was able to increase the level, of tumour necrosis factor (TNF) (TMD 100 micrograms.m-1, p < 0.05 vs control cultures), and of granulocyte macrophage colony-stimulating factor (GM-CSF) (TMD 1, 10 and 100 micrograms.ml-1 p < 0.05, < 0.05 and < 0.01, respectively, vs control cultures). In cultures of AM alone, TMD did not induce changes in the levels of any of the tested cytokines, whilst in supernatants from PBL lymphocyte cultures, TMD at 1, 10 and 100 micrograms.ml-1 increased the amounts of GM-CSF (p < 0.01 each comparison vs control cultures). Thus, TMD seems to directly stimulate lymphocytes to secrete GM-CSF and to modulate macrophage-lymphocyte interactions, resulting in the release of TNF and GM-CSF.


Relationship of upper airways disorders to FEV1 and bronchial hyperresponsiveness in an epidemiological study.

Associations of upper airways disorders (UAD) with forced expiratory volume in one second (FEV1) and bronchial methacholine response were studied, taking smoking habits into account. We used epidemiological data drawn from a population of 324 men, aged 27-58 yrs. Lower FEV1 level was related to hay fever (p = 0.01), usual (p = 0.01) and chronic (p = 0.02) rhinitis and common cold on the day of examination (p = 0.04). Allowance for the major potential confounding factor, tobacco smoking, showed similar results. Bronchial methacholine response was heightened in men reporting hay fever compared to those without (p = 0.01) but also in men reporting chronic rhinitis (p = 0.06), a group which did not exhibit skin prick test positivity more often than other subjects. Exclusion of asthmatics and taking into account smoking and skin prick test positivity yielded mostly similar results. Our data support the hypothesis of an association between lung impairment, as assessed by lower FEV1 and bronchial hyperresponsiveness to methacholine, and different types of UAD, allergic or not.


Relationship of the type of tobacco and inhalation pattern to pulmonary and total mortality.

Data from The Copenhagen City Heart Study, a prospective population study, were analysed to investigate the influence of the type of tobacco and inhalation on pulmonary and total mortality. The study sample comprised 6,511 men and 7,703 women, selected randomly after age-stratification from the general population. There were 2,986 plain cigarette smokers, 3,222 filter cigarette smokers, 1,578 smokers of cheroots/cigars, 433 male pipe smokers and 773 subjects smoking more than one type of tobacco. From 1976 until the end of 1989, 2,765 subjects died. Lung cancer was considered as main death cause in 268. Chronic obstructive pulmonary disease (COPD) was considered as the main cause in 94 cases and main or contributory cause of death in 195 cases (COPD related mortality). Current smokers had a higher risk of total mortality compared to lifetime nonsmokers: the relative risks (RR) ranged between 1.2 for male pipe smokers and 2.4 for female plain cigarette smokers. With regard to lung cancer mortality, the RR ranged between 4.1 for male pipe smokers and 7.9 for female plain cigarette smokers. Even higher RR values were estimated for COPD related mortality. In both sexes, the RR for the investigated end-points were lower in cheroot/cigar smokers and in pipe smokers than in cigarette smokers, but these differences were markedly diminished after an adjustment for the inhalation habit. The present study substantiates the view that tobacco smoking increases pulmonary and total mortality. The small differences between the various types of tobacco are probably caused by different inhalation patterns.


Influence of posture on mechanical parameters derived from respiratory impedance.

The influence of posture on respiratory mechanics was investigated in 10 healthy volunteers, using the forced pseudorandom noise technique. Subjects were studied in four randomly selected positions: sitting; sitting with the head turned sideways; supine; and prone with the head turned sideways. Respiratory compliance (Crs), inertance (Irs) and resistance estimated at 4 Hz (R4), were calculated by fitting respiratory impedance by a 4 parameter model with a frequency dependent resistance. When subjects changed from sitting to lying, whilst maintaining the head in the same position in relation to the body, Irs increased and Crs decreased, probably due to gravitational effects. R4 was significantly higher in the supine position than in either of the sitting or the prone positions. These results demonstrate that changes in lung volume cannot completely explain the influence of posture on respiratory resistance, and indicate upper airway geometry as a determinant factor of respiratory resistance.


Comparison of nebulized and sprayed topical anaesthesia for fibreoptic bronchoscopy.

We compared the efficacy of nebulized (N) and sprayed (S) topical anaesthesia prior to fibreoptic bronchoscopy in a blinded study involving 54 patients aged 57 +/- 26 yrs (mean +/- SD). Cough frequency, recorded on cassette tape, was the index of efficacy. All patients received 100 mg lignocaine sprayed into the pharynx, or nebulized in random order prior to bronchoscopy, and all received intravenous diazepam sedation. Each patient received a further 100 mg of lignocaine solution through the bronchoscope onto the vocal cords and major airways during the procedure. No significant difference was found in overall cough frequency between N and S groups (8.7 +/- 6.9 coughs.min-1 N vs 10.5 +/- 6.0 S), and cough frequency was also similar between N and S during the periods above and below the vocal cords. Furthermore, no differences were found in cough frequency between N and S among smokers, patients with asthma and COPD, and patients who had a biopsy procedure, although a trend was seen in all comparisons towards a lower cough frequency with the nebulized route. Most patients in the S group found the spray unpleasant, whereas only one in the N group complained. We conclude that nebulized and sprayed lignocaine have similar efficacy as topical anaesthetics in fibreoptic bronchoscopy, but patient preference favours the nebulized route.


The accuracy of dry gas meters at continuous and sinusoidal flows.

Dry gas meters are used in physiology laboratories to measure minute ventilation. The accuracy of these meters must be known since an error, of for instance 5%, affects the subsequent calculations for oxygen consumption by the same amount. Two precalibrated DTM-325 dry gas meters (American Meter Co., Philadelphia, PA, USA) were therefore tested for accuracy against a 350 l Collins chain-compensated gasometer. Provided that at least 25 l was passed per measurement then: a. continuous flows (air saturated with water vapour) between 60 and 150 l.min-1 were measured with an error of < 1%; and b. sinusoidal flows (ambient air) between 8 and 100 l.min-1 were misread by < 1% and the error was still within 2% at 140 l.min-1. It may, therefore, be concluded that the two precalibrated dry gas meters studied are valid volume measuring devices.


Massive pleural effusion: an unusual presentation of Castleman's disease.

Giant lymph node hyperplasia (Castleman's Disease) is a rare cause of pleural effusion. We report the case of a 51 yr old West Indian male, who presented with a recurrent massive pleural effusion, due to a tumour arising from the pleura. He underwent parietal pleurectomy and subtotal excision of the tumour. Histological analysis of the specimen showed the features of multicentric Castleman's disease. Nine months following surgery he remains well, with no recurrence of the effusion.


Chronic extrinsic allergic alveolitis in a family with idiopathic pulmonary fibrosis: the importance of histological diagnosis.

We report a patient who presented with progressive exertional dyspnoea, chronic cough and radiographic signs of interstitial lung disease. Since several of his family members were known to have familial idiopathic pulmonary fibrosis he was also suspected to suffer from this disease. After thorough investigation, including histological examination of lung biopsies obtained by thoracoscopy, a diagnosis of chronic extrinsic allergic alveolitis was made. Current knowledge of familial idiopathic pulmonary fibrosis is discussed. This case report underlines the importance of a histological diagnosis in interstitial lung disease.


Ring finger in the peroxisome assembly factor-1.

The peroxisome assembly factor-1 (PAF-1) is reported here to contain the signature subsequence for a ring finger motif in its carboxyl-terminus. This conserved subsequence in PAF-1 may be the key to a gene expression regulatory pathway important in peroxisome biogenesis.


The 5' untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells.

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5' untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5'utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5'utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3'utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5'utr was essential for the expression of GPx gene. However, the deletion of a part of the 3'utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.


Comparative analysis of sodium-dependent L-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex.

Uptake of [3H]L-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. L-trans-PDC inhibited the synaptosomal glutamate transport 2-4-fold stronger than the astroglial uptake. The substrate analogues DL-threo-beta-hydroxy-aspartate, DL-aspartate-beta-hydroxamate, L-aspartate and D-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas D-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.


Photoaffinity labeling of the phylloquinone-binding polypeptides by 2-azidoanthraquinone in photosystem I particles.

A photoaffinity label, 2-azido-9,10-anthraquinone, binds at the quinone-binding (Q phi) site with high affinity and can substitute for the secondary acceptor, phylloquinone, in photosystem I reaction center of spinach. Phylloquinone-depleted photosystem I particles reconstituted with azido[3H]anthraquinone were illuminated with UV light and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The large core polypeptides (psaA and/or psaB) were selectively labeled. The labeling was competitively inhibited in the presence of anthraquinone. These results indicate that the Q phi site is located on psaA or psaB polypeptides.


The second cholera toxin, Zot, and its plasmid-encoded and phage-encoded homologues constitute a group of putative ATPases with an altered purine NTP-binding motif.

It is shown that the second cholera toxin, Zot, ORF3 product of Pseudomonas plasmid pKB740, and ORF424 product of bacteriophage Pf1 are a group of closely related proteins containing a modified version of the purine NTP-binding motif, with a drastic substitution of tyrosine for a conserved glycine. They are distantly but reliably related to the product of gene I of filamentous bacteriophages which is a putative ATPase containing the classical NTP-binding motif and is involved in bacteriophage assembly and exit from the bacterial cell. Hydropathy analysis suggests that the Zot and gene I product may have a similar transmembrane topology. It is hypothesized that Zot may possess ATPase activity and modify the membrane structure of its target cells in an ATP-dependent fashion. Genes for Zot and the related protein of pKB740 are likely to have evolved from gene I of a Pf1-like bacteriophage.


Human liver and human placenta both contain CMP-NeuAc:Gal beta 1-->4GlcNAc-R alpha 2-->3- as well as alpha 2-->6-sialyltransferase activity.

A high pH anion exchange chromatographic (HPAEC) system for the separation of isomeric sialo-oligosaccharide products was developed. Employing this system, using Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6Man beta 1-->4GlcNAc as a substrate, a Gal beta 1-->4GlcNAc-R alpha 2-->3-sialyltransferase activity was detected for the first time in human liver. This activity is expressed together with the prevalent alpha 2-->6-sialyltransferase. Furthermore, in addition to the major alpha 2-->3-sialyltransferase, a low but distinct activity of alpha 2-->6-sialyltransferase was detected in human placenta. This activity could not be found by methods based on methylation analysis or high resolution NMR spectroscopy. It is concluded that HPAEC, in combination with the use of the pentasaccharide as an acceptor substrate, is suited for the specific detection of minor, Gal beta 1-->4GlcNAc-specific sialyltransferase activities.


Molecular cloning and developmental expression of a human kidney S-adenosylmethionine synthetase.

cDNA clones encoding the human kidney S-adenosylmethionine synthetase (kidney-type isozyme) were isolated. The amino acid sequence deduced from the cDNA indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,660 Da. The predicted amino acid sequence of this protein shares 84% similarity with that of human liver S-adenosylmethionine synthetase (liver-type isozyme). In addition, the developmental expression of these two isozyme mRNAs has been studied in the human liver using the reverse transcription-polymerase chain reaction (RT-PCR).


The thyroid hormone response element is required for activation of the growth hormone gene promoter by nicotinamide analogs.

N'-Methylnicotinamide and nicotinamide, which decreased in vitro ADP-ribosylation of nuclear proteins and/or cellular NAD+ content, selectively increased the basal expression of the rat growth hormone (GH) gene promoter and its response to triiodothyronine (T3). This increase was not found when the thyroid hormone response element (TRE) was deleted from the promoter. Transfection with an expression vector for the T3 receptor inhibited basal activity of the TRE-containing promoter and repressed the stimulatory effect of N'-methylnicotinamide. The addition of hormone relieved this inhibition and enhanced transcription above levels found in the absence of the transfected receptors. These results suggest a modulatory role of ADP-ribosylation in hormonal regulation of gene expression.


Unexpected intron location in non-vertebrate globin genes.

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.


Families of metalloendopeptidases and their relationships.

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in the other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.


Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.


Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution.

The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.


Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.


Production of an antiserum specific to the ADP-ribosylated form of elongation factor 2 from archaebacteria and eukaryotes.

An antiserum to ADP-ribosylated elongation factor 2 (ADPR-EF-2) from S. acidocaldarius was raised in rabbits using stained, homogenized, ADPR-EF-2-containing slices from SDS-gels as a source of antigen. Elongation factor 2 (EF-2) from S. acidocaldarius was cloned in E. coli and the expressed gene product was used in order to adsorb all anti-EF-2 antibodies which do not contain the ADP-ribosyl group within their epitopes, as E. coli is unable to synthesize the ADP-ribosyl acceptor diphthamide. The remaining antibodies were specific to ADP-ribosylated EF-2 from Thermoplasma acidophilum, S. acidocaldarius and Desulfurococcus mucosus. ADP-ribosylated EF-2 from eukaryotic sources also reacted with the adsorbed antiserum as shown for EF-2 isolated from the killi-fish Cynolebias whitei, the mouse species BALB/c and Han/Wistar rats. The adsorbed antiserum did not cross-react with ADP-ribosylated actin or rho protein or with FAD-containing D-amino acid oxidase.


13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.


Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.


Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli.

The two genes encoding the constituent subunits of the Thermoplasma acidophilum proteasome were expressed in Escherichia coli yielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.


Lamin A gene expression is specifically suppressed in v-src-transformed cells.

By a differential screening procedure, we have obtained several cDNAs the expression of which was down-regulated in v-src-transformed 3Y1 cells compared to parental 3Y1 cells. One of these cDNAs, termed N26, was extensively analyzed. Sequence analysis revealed that N26 cDNA was 3,095 nucleotides in length and contained an open reading frame of 665 amino acid residues. Based on an homology search at the nucleotide level, it was postulated that N26 gene encodes lamin A. The expression of the N26 gene was not suppressed in other types of transformed cells, such as v-mos-, v-ras- and SV40-transformed 3Y1 cells.


Expression of human collagen type IV genes is regulated by transcriptional and post-transcriptional mechanisms.

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two alpha 1(IV) and one alpha 2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run-on analysis and mRNAs by RNAse protection assay indicates the involvement of transcriptional as well as post-transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady-state levels alpha 1(IV)/alpha 2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post-translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.


Loss of transcription factor AP-1 DNA binding activity during lymphocyte aging in vivo.

The main feature of cellular senescence is cessation of cell proliferation. Protooncogene c-fos, which is required for the cell to enter into DNA synthesis, is repressed in senescent fibroblasts. Diminished expression of c-fos and impaired formation of AP-1, which is a complex of c-Fos and c-Jun proteins acting as a transcription factor, was found in lymphocytes derived from old (> 18 months) mice and stimulated with Con A. There were no differences in c-jun expression and formation of other transcription factors (AP-2 and AP-3) between lymphocytes isolated from old and young mice.


Characterisation of the expression and post-translational processing of the preprotachykinin-I gene and the regulated release of tachykinins by the RINm5F cell-line.

Normal transcription and postranslational processing of the preprotachykinin (PPT)-I gene and regulated release of substance P and neurokinin A by the rat pancreatic endocrine cell-line, RINm5F, has been demonstrated, using radioimmunoassays (RIAs), reversed-phase (rp)HPLC and Northern blot analysis. This is the first stable cell-line found to express the PPT-I gene and provides an opportunity for investigating PPT-I gene expression and tachykinin biosynthesis. RIN5mF cells are a model for the pancreatic beta-cell, which is not known to exhibit PPT-I gene expression which may, therefore, be a feature of the transformed state of these cells. These data may imply that the tachykinins are important in pancreatic islet embryogenesis.


Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin.

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by microM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.


Biochemical properties of rat protein kinase C-eta expressed in COS cells.

Using a PKC-epsilon cDNA probe a cDNA for PKC-eta has been cloned from a rat lung cDNA library. When expressed in COS cells, rat PKC-eta appeared as an 84 kDa protein. PKC-eta expressed in COS cells, was solubilized by 1% Triton X-100 and purified away from the endogenous PKC-alpha by ammonium sulphate fractionation. The activity of this PKC-eta preparation was characterized with respect to cofactor dependence and substrate specificity. Various PKC pseudosubstrate peptides are phosphorylated by PKC-eta in a phospholipid and TPA-dependent but calcium-independent manner. The polypeptide histone IIIS is a poor substrate.


New mouse 5-HT2-like receptor. Expression in brain, heart and intestine.

A novel member of the family of G protein-coupled receptors has been isolated from a mouse brain cDNA library by screening with polymerase chain reaction (PCR) generated fragment of mouse genomic DNA amplified using degenerated primers. Sequence comparison demonstrates that the encoded protein sequence shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from COS cells transfected with this cDNA, corresponds to a new 5-HT2-like receptor that we propose to call 5-HT2C. Its major sites of expression are in the mouse intestine and heart, also with detectable expression in brain and kidney. We speculate that it could account at least in part for the 'atypical' functions attributed to the 5-HT1C/5-HT2 receptors.


Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4).

2-Dansylamino-2-deoxy-D-galactose (GalNDns) is a useful fluorescent probe to study the interaction of non-fluorescent sugars with the B4 lectin from Vicia villosa seeds (VVLB4). Binding of the lectin to GalNDns leads to a 5.2-fold increase in Dansyl fluorescence with a concomitant 10 nm blue shift in its emission maximum. The strong binding of GalNDns (Ka = 7.33 x 10(4) M-1 at 20 degrees C) is due to a favourable entropic contribution to the association process. Among the other sugars studied, GalNAc alpha 1-O-Ser followed by Me alpha GalNAc are the best ligands. 2-Deoxygalactose, galactosamine and galactose are 2013, 469 and 130 times weaker ligands, respectively, as compared to GalNAc, whereas GalNDns is about 2.44 times more potent than GalNAc, indicating that substitutions at the C-2 position of GalNAc have a considerable influence on the binding affinities. Equatorial orientation of the hydroxyl group at C-3 and axial orientation at C-4 as in galactose are important for the interaction with VVLB4. The C-6 hydroxyl group is not indispensable. The binding site of the lectin is directed exclusively towards monosaccharides alone. Interestingly enough, despite its preference for Me alpha GalNAc over Me beta GalNAc, in oligosaccharides, the lectin prefers terminal beta-linked GalNAc as compared to the alpha-linked one.


Molluscan putative prohormone convertases: structural diversity in the central nervous system of Lymnaea stagnalis.

In the cerebral ganglia of the central nervous system of the freshwater snail Lymnaea stagnalis many neuropeptides are proteolytically processed from larger prohormones at sites marked by both single and multiple basic amino acids. In the present study we identified cloned cDNA and PCR products corresponding to three putative endoproteases that may be involved in prohormone processing. The cDNA encodes a protein of 653 residues with an overall sequence identity of 60%, 41%, 35%, 40%, and 27% with the recently characterized endoproteases PC2, PC1/3, PC4, furin and Kex2, respectively. The Lymnaea preproconvertase has approximately 80% homology with the catalytic domain, and approximately 40% and approximately 50% with the N- and C-terminal part, respectively, of the vertebrate PC2. Two cloned PCR products, Lfur 1 and Lfur 2, show highest sequence identity to furin. Expression of the LPC2 gene is exclusively in the central nervous system, where two LPC2 transcripts of 3.0 and 4.8 kb were detected.


Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant.

An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.


The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.


Primary structure required for the inhibition of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.


A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.


Gelation of liposome interior. A novel method for drug encapsulation.

Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X-Ray diffraction, electron microscopy, and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel-like phase.


The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor: a site-directed mutation study.

Wild-type and mutant serotonin 1A receptors were transiently expressed in COS-7 cells using the infection-transfection variant of the vaccinia virus/T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82-->Asn82, Asp116-->Asn116, and Ser198-->Ala198 all resulted in a decrease in affinity for 5-HT by 60-100-fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199-->Ala199, was too weak to be measured, 5-HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.


Utrophin, the autosomal homologue of dystrophin, is widely-expressed and membrane-associated in cultured cell lines.

Utrophin, the autosomal dystrophin-related protein (DRP), is expressed in HeLa cells, smooth muscle-like BC3H1 cells from mouse brain, COS monkey kidney cells, the P388D1 monocyte-macrophage cell line and untransformed human skin fibroblasts, as well as in rat C6 glioma and Schwannoma cells. It was undetectable, however, in the Sp2/O mouse myeloma cell line and in hybridoma lines derived from it. Dystrophin was not detected in any of these cell lines. Although all utrophin-containing cells were capable of forming monolayers in culture, no major effects of either attachment to substratum or length of time in culture (2-17 days) on utrophin levels were observed. After subcellular fractionation of BC3H1 or glioma cells, nearly all of the utrophin was found in the Triton-soluble fraction, suggesting an association with cell membranes.


Transport of proteins into the various subcompartments of mitochondria.

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targeting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targeting pathways to this subcompartment are known to exist.


Low translational efficiency of the F1-ATPase beta-subunit mRNA largely accounts for the decreased ATPase content in brown adipose tissue mitochondria.

The half-life of the F1-ATPase beta-subunit (F1-beta) mRNA in ATPase-poor brown adipose tissue (BAT) (t1/2 = 9.5 h) was found to be 3-7-fold shorter than in liver (t1/2 = 27 h) and heart (t1/2 = 63 h) of mice. When translated in reticulocyte lysate, a 2-3-fold lower efficiency appeared with F1-beta mRNA from BAT than from other tissues. The in vitro synthesized F1-beta protein precursors of BAT, liver and heart origin were imported and processed by mouse liver mitochondria with equal efficiency. The results indicate that the pool of abundant F1-beta mRNA in BAT is not fully translatable, most likely due to its low metabolic stability.


Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.


m-iodobenzylguanidine increases the mitochondrial Ca2+ pool in isolated hepatocytes.

The incubation of isolated hepatocytes with the inhibitor of protein mono ADP-ribosylation, m-iodobenzylguanidine (MIBG), resulted in an increase in the size of the mitochondrial Ca2+ pool, without alteration of the non-mitochondrial Ca2+ store(s). This increase was abolished when the cytosolic free Ca2+ concentration ([Ca2+]i) was buffered by prior loading of the cells with fluo 3. Elevating [Ca2+]i by releasing the endoplasmic reticular Ca2+ store with 2,5-di-(tert-butyl)-1,4-hydroquinone resulted in a synergistic increase in the magnitude of the mitochondrial Ca2+ pool. A role for protein ADP-ribosylation in the intracellular regulation of mitochondrial Ca2+ homeostasis is suggested.


Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.


Induction of cytokines in glial cells by trans activator of human T-cell lymphotropic virus type I.

Human T-cell lymphotropic virus type I (HTLV-I), the cause of adult T-cell leukemia, is also associated with the neurological disease, TSP/HAM (tropical spastic paraparesis/HTLV-I associated myelopathy). The HTLV-I genome encodes a protein, Tax, that trans activates viral and cellular gene transcription. To understand the mechanisms for the production of cytokines by HTLV-I in nervous tissue, we examined their expression in glial cells which carried the Tax-expressing vector. We demonstrated that Tax expression enhanced the production by glial cells of interleukin (IL)-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF) beta. We suggest that the excessive production of cytokines in nervous tissue may play a role in pathogenesis of TSP/HAM. Glial cells that carry the tax gene may provide a model useful for in vitro study of the mechanism of production of cytokines in the nervous system.


Cyclosporin A protects pancreatic islet cells from nitric oxide-dependent macrophage cytotoxicity.

It has been shown earlier in an in-vitro model of inflammatory islet cell death that activated macrophages lyse islet cells via the release of nitric oxide. Here we report that cyclosporin A suppresses macrophage cytotoxicity. Control experiments showed that the immunosuppressive drug does not improve the defences of islet cells against nitric oxide but inhibits the release of nitric oxide from LPS-stimulated macrophages. This property of cyclosporin A may contribute to the preservation of beta cell function seen in cyclosporin A-treated patients with recent onset type I diabetes.


Interaction of nitrogen dioxide with human plasma. Antioxidant depletion and oxidative damage.

Nitrogen dioxide (NO2.) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO2. caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of alpha-tocopherol, bilirubin and ubiquinol-10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation, alpha-tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of alpha-tocopherol. We conclude that ascorbic acid, protein -SH groups, uric acid and alpha-tocopherol may be important agents protecting against NO2. in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO2..


DNA binding of histone H1 is modulated by nucleotides.

Histone H1 acts as a general repressor of transcription in eukaryotes by organizing nucleosomes into inaccessible condensed forms of chromatin. The capability of H1 to bind to DNA with some sequence specificity is likely to be critical in the control of these processes. We show here that ATP and several other nucleotides, including non-hydrolyzable derivatives, can inhibit DNA binding of H1. The results also show that ATP differentially affects binding of H1 to DNA in a fashion enhancing nucleotide sequence specificity of the binding. The study suggests a novel mechanism of modulation of H1 activity that has important implications for the role of H1 as a transcriptional regulator.


Stilbene synthase from Scots pine (Pinus sylvestris).

Stilbene synthases are named according to their substrate preferences. By this definition, enzymes preferring cinnamoyl-CoA are pinosylvin synthases, and proteins with a preference for phenylpropionyl-CoA are dihydropinosylvin synthases. We investigated the assignment of a stilbene synthase cloned from Scots pine (Pinus sylvestris) as dihydropinosylvin synthase and the proposal of an additional pinosylvin synthase [1992, Plant Mol. Biol. 18, 489-503]. The results show that the previous interpretation was misled by several unexpected factors. Firstly, we found that the substrate preference and the activity of the plant-specific protein expressed in E. coli was influenced by bacterial factors. This was reduced by improvement of the expression system, and the subsequent kinetic analysis revealed that cinnamoyl-CoA rather than phenylpropionyl-CoA is the preferred substrate of the cloned stilbene synthase. Secondly, mixing experiments showed that extracts from P. sylvestris contain factor(s) which selectively influenced the substrate preference, i.e. the activity was reduced with phenylpropionyl-CoA, but not with cinnamoyl-CoA. This explained the apparent differences between plant extracts and the cloned enzyme expressed in E. coli. Taken together, the results indicate that the cloned enzyme is a pinosylvin synthase, and there is no evidence for a second stilbene synthase. This study cautions that factors in the natural and in new hosts may complicate the functional identification of cloned sequences.


Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Completion of the primary structure of the complex?

The amino acid sequences of two nuclear-encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N-alpha-amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear-encoded subunits of bovine complex I have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.


Photolabeling of the phosphate binding site of chloroplast coupling factor 1 with [32P]azidonitrophenyl phosphate.

Chloroplast F1-ATPase (CF1) was photolabeled by a radiolabeled photoactivatable derivative of Pi, 4-azido-2-nitrophenyl [32P]phosphate (ANPP). The radioactivity was localized in the beta subunit of CF1. Upon cleavage of the beta subunit by cyanogen bromide, the predominantly labeled peptide was recovered, which was subsequently subjected to tryptic digestion. A tryptic peptide (spanning Ile312-Arg354), was found to contain nearly all the covalently bound radioactivity. By Edman degradation, the labeled amino acid residues were identified as Tyr328, Val329 and Pro330. The labeled beta-Tyr328 of CF1 is the equivalent of beta-Tyr311 of F1 from beef heart mitochondria, which was previously found to be photolabeled by ANPP [J. Garin et al. (1989) Biochemistry 28, 1442-1448].


Primary structure of frog rhodopsin.

Amphibians have been employed extensively to study the anatomy, physiology, biochemistry, and cell biology of the visual system for decades, yet there have been no reports concerning the primary structure of amphibian visual transduction components. Thus, we have determined the entire nucleotide sequence of frog (Rana pipiens) rhodopsin cDNA, including a putative transcription start point and poly A tail, by sequence analysis of PCR products and mRNA. The open reading frame predicts an opsin of 354 residues, six residues longer than the mammalian rod opsins, containing 11 potential phosphorylation sites in the C-terminal domain. RNA blot analysis revealed two transcripts of ca. 1.7 and 3.1 kb. Frog rhodopsin exhibits approximately 85% identity to mammalian rhodopsin at the amino acid level. Sequence analysis of additional components will produce the framework from which a more detailed understanding of amphibian phototransduction can emerge.


Half-life of ubiquinone-9 in rat tissues.

The half-life of ubiquinone-9 in various rat tissues was determined. Rats were injected intraperitoneally with [3H]mevalonate and the decay of radioactivity incorporated into ubiquinone-9 was followed using reverse-phase HPLC. The half-life varied between 49 h (testis) and 125 h (kidney).


Dissociation of store release from transmembrane influx of calcium in human neutrophils.

Release of Ca2+ from intracellular stores was visualised in individual neutrophils in the presence of the Mn2+ or SKF 96365. Influx of Mn2+ quenched fura-2 close to the plasma membrane but did not quench fura-2 at the site of store release. The size and location of the 'cloud' of elevated Ca2+ was unaffected by the channel blocker SKF 96365. Furthermore, the size and location was unaffected by the presence of extracellular Ca2+. This dissociation of transmembrane influx from store release demonstrates that the entry of Ca2+ into the cytosol of neutrophils occurs directly into the cytosol and not via the store site.


Glucose decreases respiratory control ratio in EL-4 tumor cells.

EL-4 ascites thymoma cells are shown to have high aerobic glycolysis and decreased Pasteur effect. At the same time, glucose produces a much smaller inhibitory effect on cell respiration (Crabtree effect) than in Ehrlich ascites carcinoma (EAC) cells. In intact EL-4 cells, the respiratory control ratio (RCR) was found to be 6.2 with endogenous substrates and 8.0 with glutamine. Glucose decreased the RCR to 3.2, by stimulating the state 4 respiration. In rat thymocytes and EAC cells, such an effect of glucose was absent (RCR of 7.0 and 7.2, respectively). It is suggested that in EL-4 tumor cells, the high aerobic glycolysis and small Crabtree effect may be due to glucose-induced 'uncoupling' of oxidation and phosphorylation.


Platelet-derived endothelial cell growth factor. Pharmacokinetics, organ distribution and degradation after intravenous administration in rats.

Platelet-derived endothelial cell growth factor (PD-ECGF) stimulates chemotaxis of endothelial cells in vitro and has angiogenic activity in vivo. Recently PD-ECGF was shown to have thymidine phosphorylase activity. In order to study possible therapeutic applications of PD-ECGF we used a rat model to determine its pharmacokinetics and tissue distribution after intravenous injection. [125I]PD-ECGF disappeared from the plasma in a biphasic manner, with estimated distribution and elimination half-lives of 17 min and 7 h, respectively. PD-ECGF was metabolized in the liver, excreted via the bile, and not accumulated in any organ system. The stability and long half-life in the circulation, together with the specificity for endothelial cells, suggest that PD-ECGF may be useful as a therapeutic agent to stimulate re-endothelialization in vivo, or, in view of its thymidine phosphorylase activity, in chemotherapy, by decreasing the pool of available thymidine.


Codon-anticodon pairing. A model for interacting codon-anticodon duplexes located at the ribosomal A- and P-sites.

The interaction between two codon-anticodon duplexes of the ribosomal A- and P-site-bound tRNAs is the key feature of the proposed model. This interaction prohibits non-canonical base pairing at the first and second positions of the codon and controls base pairing at the third position (wobbling rules ensuing from the model are in good accord with those generated from experiments). The model is capable of predicting codon context effects. It follows from the model that modifications of the first anticodon residue of the P-site tRNA can affect the stability of the A-site duplex, and that the translation of a DNA single chain analogue of mRNA should be accompanied by non-canonical base pairing at all three positions of the codon. These predictions of the model can be subjected to experimental tests.


ATP-sensitive K(+)-channels in the human adult ventricular cardiomyocytes membrane.

Using the patch-clamp method in 27 different inside-out patches it was shown for the first time that they defined the basic parameters of the functioning of single ATP-sensitive K+ channels in the human adult ventricular cardiomyocytes membrane. At [K+]o = 140 mM single channel conductance (over the linear part of the I-V relation) reaches 100 pS. The possibility of the existence of these channels' conductance sublevels as well as the cluster character of their localization in sarcolemma is shown. The channel activity demonstrates an obvious run-down with tau at about a minute. The analysed channels possess one open and two closed states.


Transcriptional- and post-transcriptional-dependent regulation of glutathione S-transferase expression in rat hepatocytes as a function of culture conditions.

Transcriptional activity of the glutathione S-transferase (GST) alpha (subunits 1 and 2), mu (subunits 3 and 4) and pi (subunit 7) gene families has been analyzed using the nuclear 'run-on' technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co-culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co-cultured for 4 days maintain approximately 30-70% of the alpha gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co-culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the mu gene family is maintained at 40-160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4-fold) and dimethylsulphoxide (approximately 2-fold) in conventional culture and/or in co-culture. In contrast to freshly isolated hepatocytes, GST pi gene transcriptional activity is observed in conventional and co-cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST alpha, mu and pi genes in conventional and co-cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.


Altered expression of G-protein mRNA in spontaneously hypertensive rats.

We have recently demonstrated that the decreased ability of hormones, forskolin and GTP to stimulate adenylate cyclase in heart and aorta from spontaneously hypertensive rats (SHR), as compared to their age-matched Wistar-Kyoto control rats (WKY), was associated with enhanced levels of Gi- and not with Gs-regulatory proteins. In the present studies we have investigated the expression of Gi-regulatory proteins at the mRNA level by Northern blotting. Total RNA of heart ventricle and aorta from WKY and SHR was probed with radiolabeled cDNA inserts encoding Gi alpha-2 and Gi alpha-3. The Gi alpha-2 and Gi alpha-3 probes detected a message of 2-3 and 3-5 kb, respectively, in both WKY and SHR, however, the message was significantly enhanced in SHR, as compared by WKY. On the other hand the cDNA probe encoding Gs alpha detected a message of 1.8 kb in heart and aorta from both WKY and SHR, however, no difference in the levels of Gs alpha mRNA was detected in SHR and WKY tissues. These results indicate that the mRNA levels of Gi alpha-2 and Gi alpha-3 and not of Gs are overexpressed in heart and aorta from SHR, which may be responsible for the increased levels of Gi as shown earlier by immunoblotting techniques. It may be suggested that the enhanced vascular tone and impaired cardiac contractility in hypertension may partly be the consequences of increased levels of Gi in heart and aorta.


Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexta.

The amino acid sequence of the first of a family of insect cardioregulatory peptides from the tobacco hawkmoth, Manduca sexta, has been determined using a combination of Edman degradation microsequencing and mass spectroscopy. This peptide contains 9 amino acid residues and an observed mass for the monoisotopic protonated molecule of 956.4 Da. There are two cysteines at positions 3 and 9 forming a disulfide bridge and the carboxyl-terminus is amidated. The structure of this peptide, Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, is identical to a peptide recently isolated from crabs called crustacean cardioactive peptide (CCAP) and we propose that this peptide be named Manduca CCAP.


Ca(2+)-mediated interaction between negatively charged and neutral liposomes.

In the present work it is shown that large unilamellar lecithin/cholesterol liposomes are able to sequester small negatively charged liposomes in the presence of divalent cations. Evidence is presented suggesting that the sequestration occurs via the formation of membrane invaginations transformed further into intraliposomal vesicles.


A subgroup of class alpha glutathione S-transferases. Cloning of cDNA for mouse lung glutathione S-transferase GST 5.7.

A full-length cDNA clone encoding the previously purified mouse glutathione S-transferase GST 5.7 [(1991), Biochem. J. 278, 793-799] has been isolated from a mouse lung cDNA library in lambda gt11. Sequencing of the clone revealed the presence of microheterogeneity in GST 5.7. Comparison of the deduced protein sequence with other glutathione S-transferases, together with previous information available on GST 5.7, indicates that the enzyme belongs to a novel subgroup within the alpha class of glutathione S-transferases. Members of the subgroup, which also include the rat GST 8-8 and perhaps chicken GST CL3, show high sequence homology with each other, but only moderate similarity to other alpha class enzymes. They share a substrate specificity profile that resembles pi-class enzymes, and are active in the conjugation of lipid peroxidation products.


In vivo phosphorylation of the 30-kDa protein of tobacco mosaic virus.

The 30-kDa protein of tobacco mosaic virus, which is involved in cell-to-cell movement function, is phosphorylated in tobacco protoplasts. To investigate which portion of the protein is phosphorylated we inoculated several truncated 30-kDa protein mutants into protoplasts and determined whether or not those truncated proteins are phosphorylated. The results showed that amino acid residues 234-261 of the 30-kDa protein are required for this phosphorylation.


Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.


Segments of Escherichia coli genome similar to the exons of human prothymosin alpha gene.

Identification of the putative prothymosin alpha homolog in Escherichia coli cells prompted the search for a prothymosin alpha-coding gene in the E. coli genome. A set of interspersed DNA segments was identified, which match various parts of the human prothymosin alpha molecule. Their location in the E. coli genome and high degree of similarity with the appropriate regions of the human prothymosin alpha gene suggest that some kind of trans-splicing should exist in E. coli, which could be responsible for bringing these putative bacterial prothymosin alpha-coding exons together.


Oncogenes and oncoproteins as tumor markers.

A major problem in the management of patients with cancer is the lack of specific tumor markers for the early detection, the accurate prediction of biological behavior and for accurate assessment of prognosis. A new and exciting answer to this problem may now become available following the discovery of specific genes associated with malignancy. The role of such genes and their products are now being identified and their role in cancer is under intense investigation. On a clinical level, these genes and their products may allow us to improve our understanding of disease etiology and provide more precise diagnostic, prognostic and therapeutic characterization of individual tumors. This paper discusses the possibilities of using the altered expression of oncogenes and tumor suppressor genes and their products in neoplastic tissues as markers for the diagnosis and prognosis of malignant disease. These data support the view that detailed analysis of such gene expression has the potential to predict a tumor's behavior as well as the response to different treatment modalities.


Intra-operative electron beam radiotherapy and abdomino-pelvic surgery for cancer: influence on immunological parameters.

Evolution of some immunological parameters was observed during the first month in 20 patients with different abdomino-pelvic cancers who underwent surgery with intra-operative radiation therapy (IORT) (mean dose of 19.44 Gy, range 15, 25). Observed parameters before (DO-) and after procedure (DO+), on seventh (D7) and fourteenth (D14) days and fifth week (D30) were: lymphocyte count, lymphocyte subsets (CD19, CD3, CD4, CD8, CD56), natural killer (NK) activity, immunoglobulins, C3 and C4b fractions of complement, soluble receptor for interleukin 2 (sIL2-R). Results showed a decrease of circulating lymphocytes (DO-: 1189 +/- 168 cells/mm3; D7: 889 +/- 91; P = 0.011), of absolute number of CD3 lymphocytes (DO-: 785 +/- 114 cells/mm3; D7: 563 +/- 86; P = 0.025), of CD4 lymphocytes (DO-: 441 +/- 70 cells/mm3; DO+: 299 +/- 43; P = 0.013) and of CD8 lymphocytes (DO-:361 +/- 50 cells/mm3, D7:250 +/- 44; P = 0.006). All values returned towards preoperative levels by D30. Absolute number of NK cells was unchanged but NK activity was significantly diminished (effector target ratio 5:1 DO-:33 +/- 5%; DO+:44 +/- 7%; D7:18 +/- 3%; D14:21 +/- 4%; D30:25 +/- 4%). sIL2-R was significantly enhanced from D7 to D30. All these impairments are moderate and these observations provide some evidence of satisfactory tolerance to IORT for abdomino-pelvic cancers during the immediate postoperative period.


Delay in the diagnosis of breast cancer: the effect of the introduction of fine needle aspiration cytology to a breast clinic.

Fine needle aspiration cytology (FNAC) was introduced to a District General Hospital Breast Clinic in 1984 and since that time the rate of surgical excision biopsy for benign breast disease has halved. The rate of benign to malignant breast operations fell from 2.0 to 0.7 over a 7-year period from 1982 to 1988. During this period the diagnosis of breast cancer was made at the first clinic appointment in 85% of women. However, a delay in diagnosis of more than 50 days occurred in 33 patients (6.9%) and there was little difference in the annual rate of delayed diagnosis despite the addition of FNAC as an extra diagnostic tool in the later years of the study. Eighty-five per cent of patients with a delayed diagnosis were under the age of 55 (mean and median age 47) years. FNAC is a useful adjunct in the management of patients with diagnostic breast problems, but even in combination with mammography is not always sufficiently sensitive to exclude malignancy, particularly in young women.


Surgery for local recurrences following deficient radical mastectomy for breast cancer: a selected series of 39 cases.

Thirty-nine patients with locally recurrent cancer, previously treated elsewhere by mastectomy, were considered. At clinical examination, doubts arose as to the efficiency of previously performed mastectomies. Recurrent lesions, in the absence of distant metastases, were nodular, cutaneous or subcutaneous, in the area of previous 'radical mastectomy'. Second surgery consisted of a wide excision together with a surgical revision of axilla. No radiotherapy was administered to the thoracic wall after surgery. Adjuvant chemotherapy (CMF) was given to 26 node-positive women. Median follow-up was 48 months. Pathological reports showed that portions of mammary gland and axillary lymph nodes had been left behind by primary surgery in 29 and 34 cases, respectively. In 26 cases lymph nodes were metastatically involved. Local control has been maintained in 32 patients, 21 of whom are alive and free of disease.


Stage I cutaneous malignant melanoma: risk factors of loco-regional recurrence after wide local excision and clinical perspectives.

Two hundred and nineteen patients admitted to the Centre Claudius Regaud over a 14-year period for a stage I cutaneous malignant melanoma were retrospectively evaluated for loco-regional recurrence rates, risk factors and survival rates following wide primary excision. Five and 8 year survival rates corrected for deaths owing to concurrent illness were 77% and 73%. The loco-regional control rate was 69% (151/219). Distant metastases occurred in 59% (40/68) of patients who had a loco-regional recurrence, versus 11% (16/151) of patients when loco-regional control was obtained (P < 0.001). Multivariate analysis was used to ascertain which risk factors act independently as predictors of poor loco-regional control. Anatomical location of the primary ('head and neck-trunk-hands and feet' vs 'proximal limb') and thickness formed the best model in this respect among 11 prognostic factors studied. Since loco-regional recurrence may be associated with an increased risk of distant metastatic disease, we advocate the use of elective regional lymph node dissection in stage I patients at high risk of loco-regional recurrence in the hope that a portion of these patients may have increased survival owing to lack of development of widespread metastases.


Is preoperative transrectal ultrasonography of value in localised prostatic carcinoma? A blind comparative study between preoperative transrectal ultrasonography and the histopathological radical prostatectomy specimen.

Digital rectal examination (DRE) for staging is subjective and unreliable. Understaging has been reported in 25-72% and clinical overstaging of T3 tumors varies from 24-50%. In the present study of 15 patients, transrectal ultrasound (TRUS) staging was compared, in a blind comparative fashion, with pathological staging of the surgical specimen. Multifocal lesions were present in 8/15 patients (53%). A distinction was made between capsular involvement and 'clear' capsular penetration. TRUS was more sensitive in predicting capsular involvement than DRE (83% vs 17%), but the specificity was low (67% vs 100%). If capsular perforation was considered, the sensitivity and specificity of TRUS are 43% and 91%, respectively. Sensitivity and specificity for seminal vesicle tumor involvement by TRUS was 63% and 86%. Using TRUS the overall staging was improved by 33% compared with DRE and therefore TRUS is considered to be a valuable acquisition in localising and staging prostate cancer.


Radical prostatectomy for localized prostate cancer.

A retrospective analysis of 70 patients, undergoing a radical prostatectomy in 1989 and 1990 is reported. The value of computed tomography (CT) scanning in preoperative lymph node staging should be reconsidered. Evaluation of the resection margins is of utmost importance as is the distinction between capsular invasion, penetration and transgression. The etiology of local failure and its treatment are discussed.


Malignant thyroid tumours 1932 to 1972: the outcome in 492 patients.

We report on 492 patients treated for thyroid cancer at the Royal Marsden Hospital between 1932 and 1972: 380 of the patients are now dead. Eighty-three per cent (n = 410) are tumours of follicular cell origin. We found the crude 20-year probability of survival to be 54% in papillary carcinoma, 29% in follicular carcinoma and 1% in anaplastic carcinoma. Considering solely cancer deaths, the survival in papillary cases was 68%. Age, histology (tumour type), extent of primary tumour (T stage), haematogenous metastases (M1 stage) and, to a lesser extent, sex exercised a significant influence on survival. We support the use of these five factors to produce a prognostic index for estimating the level of risk for tumours of follicular cell origin. We add our experience in the non-follicular cell tumours of medullary carcinoma (3%), lymphoma (7%) and fibrosarcoma (1%).


Retroperitoneal sarcoma in a series of 51 adults.

Fifty-one patients with retroperitoneal sarcoma underwent surgery at the University Clinics of Hamburg from 1970 to 1988. Malignant schwannoma, liposarcoma and malignant fibrous histiocytoma were the most common histological types. High grade sarcomas (G3) predominated (42%), while 26% were graded G2 and 33% G1. Complete resection was possible in 59% of cases. Reoperation for local recurrence and debulking in case of not completely resectable disease was common. Regional lymph node metastasis was found in 20% of cases. The peritoneum was the preferred location of metastatic spread. All adjuvant chemotherapies were followed by tumour recurrences. After adjuvant radiotherapy three of six patients remained free of disease. In cases of residual tumour we observed few partial responses on chemo- and radiotherapy, and all patients died because of the disease. The mean survival time was 60 months, the 5- and 10-year survival rates were 35% and 15%, respectively. Factors influencing prognosis in univariate analysis included grade, size, resectability, presence of metastatic spread, histological type, microscopic local growth (infiltrative vs intact pseudocapsule of fibrous tissue) and local recurrence. With multivariate analysis grade, size and lymph node metastasis retained their prognostic significance.


How long is a 6 cm margin of resection in the stomach?

In order to assess whether the gastric wall undergoes some change in length during gastrectomy operation for cancer, we measured the variation in length of the anterior gastric wall in 25 patients. The first measurement was made at the beginning of laparotomy by placing two stitches on the anterior gastric wall and registering the distance between them. A second and a third measurement were recorded when the stomach was fully isolated just before its transection and subsequently on the anatomic table. The results indicate that the usual recommendations made by pathologists to maintain a 6 cm margin of tissue clearance proximally to the cephalic edge of the tumour, can be safely followed by the surgeons who can correctly assess, during operation, the distance between tumour and the desirable line of transection since no misleading reduction in size of the resected specimen takes place.


Saphenous vein grafts for anatomical variations encountered at surgical insertion of a hepatic artery catheter.

Fifty-five consecutive patients, with colorectal metastases confined to the liver, underwent surgical placement of a hepatic artery catheter. At angiography, abnormal hepatic arterial anatomy was present in 33% of patients. In the majority of patients, the hepatic artery catheter was inserted in the conventional manner. In eleven patients with unusual arterial anatomy, a saphenous vein graft was used to create a conduit for the catheter. Satisfactory perfusion was obtained in all patients.


Prognosis of surgically treated radiation-induced damage to the intestine.

A series of 88 patients operated on during 24 years for radiation-induced damage (RID) to the intestinal tract were retrospectively reviewed and clinical and surgical factors were related to the ultimate prognosis by multivariate analysis. The first operation was performed on the small intestine in 47 patients, the large intestine in 32 patients or both in nine patients. Postoperative complications occurred in 35 patients (40%), with fatal outcome in 12 (13%). Thirty-one patients (35%) required further surgery and altogether 19 patients (22%) ultimately died from RID. Negative prognostic factors after the first operation were postoperative intestinal leak (P < 0.05) and operation for fistula or perforation (P < 0.01). The outcome after the last operation was negatively influenced by intestinal leak (P < 0.001) by the choice of bypass as operative procedure (P < 0.01) and by operation for fistula or perforation (P < 0.01). In addition, 43% of the patients in whom the disease had progressed between two explorations died from RID. Thus, the severity of the RID as diagnosed at laparotomy, and progression of the disease between two subsequent explorations were related to the prognosis. Care should be taken to avoid intestinal leak. Resections should be preferred to bypass of injured intestine whenever possible.


Carcinoma of the pancreas and papilla of Vater--assessment of resectability and factors influencing resectability in stage I carcinomas. A prospective multicentre trial in 472 patients.

Between 1984 and 1987, 472 patients with histologically or cytologically verified carcinomas of the pancreas or papilla of Vater, were accrued in the Norwegian Pancreatic Cancer Trial. Surgical assessment revealed resectability in 29% (94 of 330) of the pancreatic tumours and 89% (25 of 28) of the papillar tumours. Tumours of the pancreatic head were resectable in 32% (84 of 259). The sensitivities of the different diagnostic methods in patients with resectable tumours were: FNAC (fine needle aspiration cytology) 80%, ERCP (endoscopic retrograde cholangio-pancreatography) 78%, PTC (percutaneous transhepatic cholangiography) 73%, ERCP with duct cytology 67%, CT (computed tomography) 58%, US (ultrasound) 42% and angiography 22%. The positive predictive values (PV+) in resectable disease were: US 29%, CT 35%, ERCP 43% and angiography 44%. Corresponding figures for unresectable disease were US 95%, CT 97%, ERCP 75% and angiography 88%. Resectable tumours of the pancreas and papilla of Vater had an average macroscopic diameter of 3.2 x 3.4 cm and 2.2 x 2.3 cm, respectively. Tumour size increased with stage. Increasing tumour size and abdominal pain combined with short diagnostic delay both decreased resectability rate, whereas a combination of long diagnostic delay and abdominal pain had a more favorable resectability rate. Radical pancreatic surgery, if effective in the treatment of carcinoma of the pancreas or papilla of Vater, should not be undertaken if any preoperative diagnostic test demonstrates signs of indisputable unresectability. Available methods for the evaluation of resectability in patients lacking such signs are insufficient. This necessitates exploratory laparotomy in many patients.


The prognostic value of the tumour markers CA 195 and CEA in patients with adenocarcinoma of the pancreas.

The serum levels of CA 195 were determined in 52 patients with histologically proven pancreatic carcinoma and compared with carcino-embryonic antigen (CEA), serum bilirubin and albumin. CA 195 levels were raised above the upper limit of 20 U/ml in 42 cases, giving a sensitivity of 80% for the detection of pancreatic carcinoma, whereas CEA was raised in only 55%. The levels of CA 195 and CEA were significantly higher in patients with metastatic disease, but potentially curable cases were not discriminated. Bilirubin and albumin levels were not significantly related to either the presence of metastases or the levels of the tumour markers. At the time of initial presentation, levels of both tumour markers correlated with the eventual duration of survival, but bilirubin and albumin did not. Significant increases in CEA and CA 195 were found in sequential blood samples, as the disease progressed. Neither CA 195 nor CEA was of sufficient sensitivity to be of value for screening, but both give an indication of the presence of metastases and of the subsequent duration of survival. CA 195 appeared to be more sensitive and might help to assess progress of the disease.


Spontaneous remission of bladder neoplasm.

A case of spontaneous remission of bladder cancer in an 85-year-old man is reported. The clinicopathological features are presented and the literature reviewed.


Puberty and polycystic ovarian syndrome: the insulin/insulin-like growth factor I hypothesis.

To provide an up-to-date review of studies that have examined the physiological effects of insulin and insulin-like growth factor I (IGF-I) on ovarian growth, maturation, and steroid synthesis, their physiological role in puberty, and their pathophysiological role in polycystic ovarian syndrome (PCOS). To deduce from these data a hypothesis, explaining the pathogenetic connections between puberty and PCOS. The most relevant studies related to this topic have been identified through a computerized bibliographic search (MEDLINE) and through manual scanning of what has been published during recent years in the most important journals in the field of reproductive endocrinology. Insulin and IGF-I stimulate ovarian growth and potentiate the actions of gonadotropins on ovarian steroid synthesis. Insulin also augments the bioactive concentrations of IGF-I and androgens through regulation of the synthesis of their respective binding proteins insulin-like growth factor-1 binding protein (IGFBP-1) and sex hormone-binding globulin (SHBG) in the liver. Insulin and IGF-I might also be able to increase the adrenal sensitivity to adrenocorticotropic hormone (ACTH). Insulin resistance with compensating hyperinsulinism is a common feature of PCOS. It is also a normal phenomenon during puberty. Polycystic ovarian syndrome often develops during puberty. Ultrasonographic investigations suggest that it is much more common during adolescence than generally assumed. Actually, there is a striking resemblance between the endocrine characteristics of puberty and some forms of PCOS. Both conditions are characterized by insulin resistance, hyperpulsatile gonadotropin secretion, hyperactive ovarian and adrenal androgen synthesis, and decreased levels of IGFBP-1 and SHBG. We propose the progressively increasing insulin levels and IGF-I activity during puberty as inducing factors in the development of PCOS in susceptible subjects.


Ovulation induction in women age 40 and older: the importance of basal follicle-stimulating hormone level and chronological age.

To determine pregnancy and livebirth rates for women age 40 and older undergoing ovulation induction and to assess the impact of basal follicle-stimulating hormone (FSH) on outcome in these patients. Prospective, observational. Fertility service of university medical center. Infertile couples in whom the female partner was age 40 or older referred for ovulation induction therapy. Assessment of basal hormonal status; ovulation induction. Clinical pregnancy rate (PR), livebirth rate. Analysis of 402 cycles in 85 women age 40 and older demonstrated a clinical PR of 3.5% per cycle (95% confidence interval [CI] 1.7% to 5.3%). The livebirth rate was 1.2% per cycle (95% CI 0.1% to 2.3%). Women with a basal FSH < 25 IU/L and age < 44 years had a clinical PR of 5.2% per cycle (95% CI 2.5% to 7.9%) compared with 0.0% per cycle (95% CI 0.0% to 2.1%) in cases in which either basal FSH was > or = 25 IU/L or age was > or = 44 (P < 0.005). The prognostic importance of basal FSH and chronological age was confirmed by multivariate logistic regression analysis. The predictive value of the resulting regression equation was high (R2 = 0.94; P < 0.01). Pregnancy and livebirth rates are generally low during ovulation induction in women age 40 and older. In combination, basal FSH and chronological age are accurate predictors of PR, in these couples and can define a subset of patients with a more favorable prognosis. The spontaneous abortion rate in women who do conceive is high, substantially lowering the livebirth rate.