Spatial disorientation in general aviation accidents. Spatial disorientation (SD) was the third highest "cause" of fatal accidents in small, fixed-wing aircraft and closely related to the second highest "cause"--"continued VFR flight into adverse weather." SD was a cause or factor in 16% of all fatal accidents. When SD was ascribed as a cause or factor in an accident, 90% of the time that accident involved fatalities. Small, fixed-wing aircraft under 12,500 lb (570 kg) accounted for 97.3% of all SD accidents. Inclement weather was associated with 42% of all fatal accidents, and SD was a cause or factor in 35.6% of these. Flight was initiated into and continued into adverse weather in 19.7 and 68.7%, respectively, of SD weather-related fatal accidents. Fog (56.8%) and rain (41.8%) were the most prevalent adverse weather conditions. These and other data attest to the importance of this psychophysiological phenomenon in flight safety.


Structural changes associated with type-A influenza in Mallard duck tracheal organ culture. Mallard duck tracheal organ cultures were used to study structural changes associated with infection with type-A influenza (A/Turkey/WIS/68) (H9N2) at the light-microscope and electron-microscope levels. Light-microscope changes in infected organ culture were cytoplasmic vacuolization, nuclear swelling, reduction in ciliated epithelium, and sloughing of epithelial cells. Ultrastructural changes included the loss of cilia and microvilli, distortion and swelling of cellular organelles, breakdown of intercellular junction, and apparent phagocytosis of the ciliated epithelium. Numerous budding virions were noted at the plasmalemma. Virus was detected by egg inoculation from all experimental cultures throughout the 192-hour experiment.


Hypoxia survival variations in male and female mice as functions of chronological and environmental factors. The inhalation by mice for 20 min of 5.5% oxygen in nitrogen, performed 29 times over 40 months, causes a mortality of 49.81%. Correlation coefficients were calculated between hypoxia mortality and different parameters: environmental--lighting, temperature, hygrometry, barometric pressure, biological--sex, age, body weight; and chronological--circadian, circannual, pluriannual. Partial correlation coefficients eliminate several interrelationships and finally point out the statistical significance of transfer from dark to light (p less than 0.001), of circadian (p less than 0.05), and of circannual (p less than 0.01) hypoxic mortality variations. Moreover, a significant (p less than 0.001) sex-related difference of mortality (males: 56.94%; females : 41.14%) was observed, independently of the environmental and chronological parameters studied.


Effect of long-term hyperbaric stress on ammonia metabolism in humans. A significant increase in blood urea was found in 11 U.S. Navy divers during 8 d of air saturation hyperbaric exposure. Similar increases in blood urea have been found in animals that convulsed during hyperbaric oxygen exposure. Therefore, it is suggested that careful attention be given to blood ammonia and urea levels in humans during long-term hyperbaric exposure.


Task-specific simulator pretraining and in-flight stress of student pilots. Student pilots (n = 20) were assigned to experimental and control groups. The experimental group received 80 min of high-fidelity, task-specific simulation prior to exposure to the initial power-on stall and spin recovery lesson unit in T-37 pilot training. Both experimental and control groups received orientation simulator experience not related to the aircraft spin series. Catecholamine excretion patterns indicated that the initial power-on stall and spin recovery lesson unit resulted in a pronounced stress response in both groups. A statistically significant difference in the norepinephrine/epinephrine ratio was found to exist between the experimental and control groups. It was concluded that task-specific simulator pretraining resulted in an altered stress response characterized by lower arousal and greater mental work than was evidenced in the control group.


Intangibles facilitating or inhibiting health care delivery systems. Proponents of general systems theory believed its principles were applicable to a variety of living systems. General systems theory was applied to health care delivery systems and provided a conceptual framework for operation. Intangibles in the health care delivery system may inhibit or facilitate open or closed systems and thereby directly or indirectly influence the quality and quantity of care. This paper reviews and discusses: 1) Basic concepts of general systems theory; 2) Application of general systems theory to health care delivery systems, including the Air Evac system; 3) Intangibles in systems which facilitate or inhibit health care delivery; and 4) Suggestions to facilitate open systems and the delivery of quality care.


Longevity in military pilots: 37-year followup of the Navy's "1000 aviators". The 37-year nonmilitary mortality rate for initially healthy aviators was determined in a followup program on the U.S. Navy's "1000 Aviator" cohort. Of the 800 survivors of World War II and the Korean conflict, 95 were found to have died from nonmilitary causes over this followup period. This is markedly less than the 208 that would be expected from a random sample of white American men over a similar period (p less than 0.005). It is also significantly less than the 143 that would have been expected from a group of men who had passed an initial insurance physical (p less than 0.005). Lower-than-expected death rates occurred in all three major categories of cause of death in this age group: cardiovascular, neoplastic, and accidental. The generally good socioeconomic background, the positive genetic influence of long-lived parents, the above average intelligence, and the health and fitness orientation of the military aviator are all thought to be factors contributing to this increased longevity.


Altitude tolerance of general aviation pilots with normal or partially impaired spirometric function. The altitude tolerance of 10 spirometrically impaired (SI) general aviation pilots with an average forced midexpiratory flow (FEF25--75%) value of 65.1% was compared to that of 10 spirometrically normal (SN) pilots. Cardiorespiratory parameters assessed at ground level (GL) and at 8,000- and 12,500-ft altitudes were blood pressure, pulmonary ventilation, oxyhemoglobin saturation, temporal artery flow velocity, heart rate, and single-lead electrocardiogram. Although altitude exposure quantitatively displaced the SI group more than the SN group, the differences were not statistically significant at the probability level of 0.05. Unifocal premature ventricular contractions were present at GL in three of the pilots and showed no further changes at altitude. Therefore, the mean FEF25--75%, values of 65% of predicted normal for the SI group becomes a reasonable option as an objective screening norm for acceptable tolerance to general aviation altitudes in the ambient-air-breathing range.


Retrograde coronary blood flow after mannitol in intact anesthetized dogs. In control dogs mannitol produced a moderate increase in coronary blood flow, a decrease in coronary vascular resistance, and a moderate increase in cardiac output. In dogs with coronary occlusion mannitol increased retrograde coronary blood flow (+25%) and collateral conductance suggesting increased blood availability to ischemic myocardium.


[Effect of X-ray contrast medium on peripheral circulation, arterial blood flow and venous capacity in the lower extremities during phlebography (author's transl)]. The influence of contrast medium (20 ml Meglumine Diatrizoate 65%; Angiografin) on peripheral circulation was studied after injection into the vena saphena magna in 12 patients (mean age 40.5 years) with markedly varicose veins. Pneumatic segment-plethysmography and "mercury-in-rubber" strain-gauge-plethysmography were used to investigate local and systemic effects of contrast medium on intravascular basic volume and pressure-dependent venous capacity. Heart rate was controlled by Ecg-monitoring, venous blood pressure by means of a Schwarzer pressure transducer, and peripheral blood flow by means of pneumatic segment-plethysmography. In addition peripheral vascular resistance was calculated. Local effect of contrast medium on large veins was a small insignificant increase (2.2%) of venous capacity. However, there was a significant increase of additional 10% after recirculation (2 P less than 0.01). Intravascular basic volume showed a significant increase as well (2 P less than 0.01). In addition the calculated increase of total intravascular volume--represented by intravascular basic volume plus venous capacity--was 16.3% related to an intravenous pressure of 70 mm Hg. According to these findings, 20 ml contrast medium enlarges the blood volume dumping into the legs with postural changes by approximately 270 ml. Arterial inflow increased significantly (2P less than 0.01) by 40% after contrast injection, whereas mean arterial blood pressure decreased simultaneously. Consequently a reduction of peripheral vascular resistance of 32% was calculated. The neglectable local effect of contrast medium on pressure-dependent venous capacity is probably due to the markedly reduced distensibility of varicose veins. In contrast a striking effect of contrast medium is observed after recirculation, which is felt to be due to a general vasodilatation of capillary vessels and pre- and postcapillary resistance vessels.


Left ventricular volumes and functioning of the reindeer heart. Left ventricular volumes were measured by cineangiocardiography in 56 sessions on 25 reindeer, together with determinations of arterial pressure and blood oxygen saturation. The heart rate with the animal kept lying on the sternum at rest was 50 b/min, the aortic blood oxygen saturation 94 to 98% and the aortic blood pressure 153/130/115 mm Hg. The left ventricular end-diastolic volume was 3.4 ml/kg of b.w., stroke volume 2.6 ml/kg, cardiac output 133 ml/kg . min, and ejection fraction 76%. If the animal was placed on its side the aortic blood oxygen saturation decreased by 2 to 3 per cent, end-diastolic volume rose 10%, stroke volume 25%, cardiac output nearly 30% and ejection fraction 14%. The heart rate and cardiac output increased in hypoxemia, but the end-diastolic and stroke volumes simultaneously decreased. If the aortic blood pressure rose during hypoxia, the end-diastolic and stroke volumes increased, while the ejection fraction first increased and then decreased again. In normoxia a rise in aortic blood pressure was associated with decreased end-diastolic and stroke volumes and ejection fraction. An increased heart rate at rest was followed by increased cardiac output, but decreased end-diastolic and stroke volumes and ejection fraction.


Preload dependence of dP/dt max, VCE max and calculated V max compared to the inotropic sensitivity of these indices of cardiac contractility. In five canine heart-lung preparations the preload dependence of dP/dt max, V max, VCE max and (dP/dt:P-EDP + C) max was related to the inotropic sensitivity of these indices. V max and (dP/dt:P-EDP + C) max are less affected by preload changes than dP/dt max and therefore should be more suitable for inotropic measurements. But even if the preload changes caused by inotropic interventions are not taken into account, V max shows the same inotropic sensitivity as dP/dt max when the contractile state is reduced and an even lower sensitivity when the contractile state is enhanced. The sensitivity of (dP/dt:P-EDP + C) max, however, is much lower over the whole range of tested inotropic states. If enddiastolic pressure is kept constant during inotropic interventions, dP/dt max is a much more sensitive inotropic index than V max. VCE max usually shows a negative correlation to the diastolic filling of the heart. In the improved inotropic state this correlation can become positive, thus making it impossible to evaluate the influence of the preload dependence on the result. None of the tested parameters shows a higher inotropic sensitivity than dP/dt max, though the preload dependence of dP/dt max is more evident. Therefore, none of these more complicated parameters has any advantage over the simple measure dP/dt max for evaluation of acute changes in contractility.


[Systolic time intervals. II. Relationship of values of children with heart diseases (author's transl)]. For 219 children between 3 and 15 years with the most frequent congenital and acquired heart failures it was investigated in what way the STI differ consistently from the corresponding standard values. It was found that all the STI of children with cardiovascular diseases can vary from the normal values scaled by heart rate even in disorders of the right heart. The STI naturally have the greatest diagnostical significance in the evaluation of failures of the left heart, especially aortic stenosis (shortened PEP lengthened QS2 and LVET), and of heart partition defects (shortened LVET, prolonged PEP, normal QS2). Statistically individual groups of heart disorders can be differentiated from one another by the STI rather well.


Amino acid sequences of alpha-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment. The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool.


Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Statistical analysis. The distribution of the different types of amino acid residue within two helical segments isolated from the low-sulphur fraction of wool keratin was examined for periodicity and larger sequence repeats. Both were detected, the former corresponding to the geometry of the proposed coiled=coil structure and the latter suggesting a distant gene duplication, though the existence of the repeat might equally well be related to interactions within the microfibril.


Influence of the peptide-chain length on disulphide-bond formation in neurohypophysial hormones and analogues. (8-Arginine)vasopressin, (8-arginine)vasotocin, oxytocin and oxypressin, the 'ring' derivatives pressinamide and tocinamide, and the extended-chain analogues Pro-Arg-Val-(8-arginine)vasopressin and (8-arginine)vasopressinoyl-Ala-Met-Ala-NH(2), were synthesized by the solid-phase method and purified by sequential gel filtration on Sephadex G-15 in 50% acetic acid and 0.2M-acetic acid. Controlled oxidation of the thiol groups of the reduced peptides obtained after deprotection with sodium in liquid ammonia gave rise to products that depended on the length of the peptide chain: (i) nonapeptides gave monomer and dimer species, (ii) hexapeptides produced mixtures containing higher polymers, and (iii) dodecapeptides gave predominantly monomer with some dimerized material. The evidence suggests that the presence of the acyclic tail tripeptide in the nonapeptide hormones induces a conformation in the preceding hexapeptide that favours the formation of an intramolecular disulphide bond. For (8-arginine)vasopressin, intramolecular disulphide-bond formation is enhanced by extension of the peptide chain from either the N- or the C-terminus. The possible significance of these studies to neurohypophysial hormone-prohormone relationships is discussed.


Magnetic-circular-dichroism studies of haem a and its derivatives. The low-temperature m.c.d.(magnetic-circular-dichroism) spectra of haem a derivatives are presented. It is shown that reduced haem a in pyridine contains the low-spin ferrous ion and that oxidized haem a in the presence of excess imidazole has the low-spin ferric form. Both the m.c.d. and the absorption spectra of haem a in excess pyridine are changed by the addition of excess sodium dithionite into a form similar to that of haems b and c. This is interpreted in terms of an attack, by reduced pyridine, at the 8-formyl group of the haem a ring. This leads to a partial assignment of the electronic spectrum of haem a.


Secretion of beta-N-acetylglucosaminidase isoenzymes by normal human fibroblasts. 1. Secretion of the lysosomal enzyme beta-N-acetylglucosaminidase (EC 3.2.1.30) by normal human fibroblast cultures was linear with respect to time up to 96h. 2. Two forms of the A isoenzyme of beta-N-acetylglucosaminidase were found in the culture medium. One form was similar to the isoenzyme found in other extracellular fluids, such as plasma and tears, the other resembled the intracellular (lysosomal) enzyme. The presence of the two isoenzymes in the culture medium appears to reflect two distinct secretory processes. 3. It is suggested that plasma acid hydrolases may be destined for incorporation into lysosomes in a manner analogous to that described for the packaging of lysosomal enzymes by fibroblasts.


The amino acid sequence of ferredoxin from the red alga Porphyra umbilicalis. The amino acid sequence of the ferrodoxin of Porphyra umbilicalis was determined by the dansyl-phenyl isothiocyanate method, on peptides obtained by tryptic, chymotryptic and thermolytic digestion of the protein or its CNBr-cleavage fragments. The molecule consists of 98 residues, has an unblocked N-terminus and shows considerable similarity with other plant-type ferredoxins. It is the first reported sequence of a red-algal ferredoxin.


Comparative study of the glycosphingolipids of chicken bursa of Fabricius and of chicken, rat and human thymus. 1. The glycosphingolipid compositions of the thymus and bursa of Fabricius of young male chickens were compared. The two tissues were found to contain complex mixtures of both neutral glycosphingolipids and gangliosides. Both tissues contained mono-, di-, tri-, tetra- and penta-glycosylceramides; the pentaglycosylceramide displayed a reaction of identity with authentic Forssman antigen when tested against a specific anti-(Forssman antigen) serum. The ganglioside G(m3) containing N-acetylneuraminic acid was the principle ganglioside of both tissues. 2. The thymus contained appreciable amounts of the simple ganglioside N-acetylneuraminylgalactosylceramide, a compound not found in the bursa. The ganglioside G(d3) (disialohaematoside) was detected in both tissues. 3. Rat and human thymus, like sheep thymus (Narasimhan, Hay, Greaves & Murray (1976) Biochim, Biophys. Acta 431, 578-591), both contained a tetraglycosylceramide species as their most complex neutral glycosphingolipid and possessed little or no Forssman antigen. They also contained a complex mixture of gangliosides. 4. The possible significance of these results is briefly discussed.


Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase. Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.


Studies on the tissue specificity of the high-mobility-group non-histone chromosomal proteins from calf. The high-mobility-group (HMG) non-histone chromosomal proteins from calf thymus, liver, spleen and kidney were extracted, and fractionated by CM-Sephadex chromatography and trichloroacetic acid precipitation. The isolated proteins HMG 1, HMG 2 and HMG 17 from the tissues were compared by polyacrylamide-gel electrophoresis, isoelectric focusing and amino acid analysis. The results show that the three proteins are very similar in the tissues studied, implying a lack of tissue specificity.


The iron-binding properties of hen ovotransferrin. 1. The distribution of iron between the two iron-binding sites in partially saturated ovotransferrin was studied by labelling with 55Fe and 59Fe and by gel electrophoresis in a urea-containing buffer. 2. When iron is added in the form of chelate complexes at alkaline pH, binding occurs preferentially at the N-terminal binding site. In acid, binding occurs preferentially at the C-terminal site. 3. When simple iron donors (ferric and ferrous salts) are used the metal is distributed at random between the binding sites, as judged by the gel-electrophoresis method. The double-isotope method shows a preference of ferrous salts for the N-terminal site. 4. Quantitative treatment of the results of double-isotope labelling suggests that in the binding of iron to ovotransferrin at alkaline pH co-operative interactions between the sites occur. These interactions are apparently absent in the displacement of copper and in the binding of iron at acid pH.


Hyaluronic acid in the pulmonary secretions of patients with asthma. Hyaluronic acid was the only glycosaminoglycan found in the pulmonary secretions of patients with asthma. The compound had a hexuronate/hexosamine molar ratio of about 1:1. Glucosamine constituted over 98% of the hexosamines, the remaining being galactosamine. The compound moved as a single spot with the mobility of standard hyaluronic acid on cellulose acetate electrophoresis, and this spot disappeared after digestion with testicular hyaluronidase. Even after extensive proteolysis and purification, the compound was associated with small amounts of protein, the major amino acids of which were aspartic acid, threonine, serine, glutamic acid, glycine and valine.


The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus. 1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5 X 10(6), which is halved to 0.76 X 10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.


Crystal structures of egg-white lysozyme of hen in acetate-free medium and of lysozyme complexes with N-acetylglucosamine and beta-methyl N-acetylglucosaminide. The binding of beta-methyl N-acetylglucosaminide (betaMeGlcNAc) to egg-white lysozyme of hen in the tetragonal crystal form was studied by X-ray diffraction techniques to a resolution of 0.25 nm. The binding of the beta-methyl glycoside is almost identical with the binding of beta-N-acetylglucosamine (betaGlcNAc). Real-space refinement of the lysozyme-alpha/beta GlcNAc and lysozyme-betaMeGlcNAc complexes allowed preliminary analysis of the conformational changes observed on binding monosaccharide inhibitors, specially in the region involving tryptophan-62 and residues 70--76. Tetagonal lysozyme crystals, grown in the absence of acetate ions, were examined by X-ray diffraction to 0.25nm resolution. The resulting difference Fourier synthesis shows no firm evidence for bound acetate ions and indicates only minor conformational changes in the side-chain positions of aspartic acid-101 and asparagine-103. The close similarity of the lysozyme structures in the presence and absence of acetate is contrary to expectations from previous n.m.r. studies.


A structural model for desmosine cross-linked peptides. Desmosine-enriched peptides were isolated from a thermolysin digest of bovine ligamentum nuchae elastin and a partial sequence was determined. A 'two-cross-link' model is proposed in which a second cross-link, perhaps lysinonorleucine, joins two peptide chains approx. 35 amino acid residues removed from the desmosine. Implied in this model is a certain asymmetry or directionality which places restrictions on the 'sense' of the peptide chains (either always parallel or anti-parallel) in order to align the cross-linking sites. Imposing such restrictions raises the possibility of specific alignment of elastin precursor molecules by microfibrillar proteins and/or aligning peptides on the precursor molecules themselves.


Incorporation of [3H]leucine into an actin-like protein in response to 1,25-dihydroxycholecalciferol in chick intestinal brush borders. 1,25-Dihydroxycholecalciferol very rapidly stimulates the incorporation of [4,5-3H]leucine into at least two proteins of the chick intestinal mucosal cells. The smaller of the two proteins has mol.wt. approx. 42000, very similar to actin. Other properties of this protein were investigated, including its solubility in salt solution and its behaviour on gel electrophoresis and isoelectric focusing, and in all cases the 1,25-dihydroxycholecalciferol-stimulated protein was indistinguishable from intestinal actin. It was also shown that the brush borders of mucosal cells contain both beta- and gamma-actin in approximately equal amounts and that both forms of the protein appear to be affected by the hormone. It is concluded therefore that one of the earliest actions of 1,25-dihydroxycholecalciferol is to stimulate the incorporation of leucine into beta- and gamma-actin of the mucosal cells or into two proteins very like them.


Separation and partial characterization of guinea-pig caseins. 1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.


Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries. 1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, alpha-subunit 40600, beta-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the alpha-subunit of the decarboxylase. 5. The molar ratio of phosphate to alpha-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [(32)P]phosphate complex by using methyl [1-(14)C]acetimidate, followed by separation of alpha-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two alpha-chains (i.e. one phosphate group/alpha(2)beta(2) tetramer). Complete phosphorylation corresponded to three phosphate groups per alpha(2)beta(2) tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 alpha-subunits:4 beta-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.


The direct determination of porphyrin carboxylic acids. High-pressure liquid chromatography with solvent systems containing phase-transfer agents. A novel method for separating porphyrin polycarboxylic acids is described and illustrated by its application to the direct analysis of biological (deep-sea medusae), clinical (urine) and chemical ('haematoporphyrin derivative') samples.


Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase. 6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.


The mechanism of haem catabolism. A study of haem breakdown in spleen microsomal fraction and in a model system by 18O labelling and metal substitution. The mechanism of bile-pigment formation from haem breakdown was studied by using 18O labelling of the molecular oxygen required for macrocyclic ring cleavage. For haem degradation by the spleen microsomal haem oxygenase system, mass spectrometry of the product bilirubin revealed that cleavage occurred by the Two-Molecule Mechanism, i.e. the terminal lactam oxygen atoms in bilirubin were derived from two different oxygen molecules. Similarly, degradation of myoglobin by coupled oxidation with ascorbate and oxygen proceeded via the Two-Molecule Mechanism. Cobalt and manganese complexes of protoporphyrin IX were not degraded by either the haem oxygenase system or the coupled oxidation system. This result suggests that the iron atom possesses unique properties in facilitating porphyrin breakdown.


Regulation of pyruvate dehydrogenase activity in rat epididymal fat-pads and isolated adipocytes by adrenaline. 1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.


Ontogeny of the vitellogenic response to oestradiol and of the soluble nuclear oestrogen receptor in embryonic-chick liver. A specific high-affinity oestradiol-binding protein was characterized in salt extracts of liver nuclei of the developing chick embryo. It is present in very small amounts at day 10 of development and is marginally stimulated by oestradiol injection into the yolk sac on day 8. Injection of oestradiol on day 10 evokes a substantial increase in the nuclear oestradiol-binding activity measured on day 12 of development. This oestradiol-binding protein has properties of sedimentation, hormone specificity and high-affinity binding very similar to those of the soluble nuclear receptor in hatched chicks. Livers from the 12-day embryos injected 48 h earlier with oestradiol do not synthesize vitellogenin, as judged by a specific immunochemical and electrophoretic assay for this oestrogen-induced protein. Traces of vitellogenin synthesis can be induced in 13-day-embryo liver, and a substantial response, equivalent to that in hatched chicks, is seen in liver from 15-day embryos injected on day 13. The development of the ability of oestradiol to increase the concentration of the soluble nuclear receptor appears to be one, but not the only, critical factor involved in the development of the ability of chick liver to synthesize vitellogenin.


A method for the analysis of protein turnover characteristics. Indirect estimation of rates of protein degradation. A method for the calculation of rate constants of degradation of a specific protein during a period of change in protein amount is described. The calculation uses measurements of rates of synthesis and amount of the protein, together with one estimate of the protein half-life. The method is demonstrated by using data from measurements made on the accumulation of fatty acid synthetase in hormonally treated explants of mammary gland from mid-pregnant rabbits, and data from other authors. These calculations demonstrate a regulated programme of co-ordinated changes in rates of synthesis and degradation during a period of change in protein mass. The role of such changes in protein degradation during protein accumulation is discussed.


Fatty acid biosynthesis in the leaves of barley, wheat and pea. 1. The incorporation of radioactivity from [1-14C]acetate into the leaf lipids of barley, pea and wheat has been studied in pulse-labelling experiments. 2. There was little increase in the total labelling of lipids after the leaves were transferred to non-radioactive medium. However, there was an increase in the relative labelling of unsaturated fatty acids. In addition, there was an increase in the relative labelling of diacylgalactosylglycerol. 3. The principal radioactively labelled acyl lipids were diacylgalactosylglycerol and phosphatidylcholine. Phosphatidylcholine showed a decreasing proportion of [14C]oleate and an increasing amount of [14C]linoleate with time. Diacylgalactosylglycerol also had decreasing amounts of [14C]oleate but, in addition, had an increasing proportion of [14C]linolenate with time. 4. The absence of significant amounts of [14C]linolenate in phosphatidylcholine appeared to exclude a role for this phospholipid in linoleate desaturation. 5. The specific radioactivities of oleate and linoleate in phosphatidylcholine, diacylgalactosylglycerol and diacylgalabiosylglycerol were very similar in any single experiment. It was concluded that these fatty acids can rapidly exchange between the three intact lipids.


Distinction between binding and endocytosis of human asialo-transferrin by the rat liver. The ability of the rat liver to bind and endocytose human asialo-transferrin was investigated in vivo. Asialo-transferrin was separated from incompletely desialylated transferrin and neuraminidase by chromatography before being labelled with (125)I. Plasma radioactivity curves and hepatic radioactivity contents measured over a 1270-fold dose range led to the following observation. At the lowest dose (0.4mug/100g body wt.), the distribution of asialo-transferrin between plasma and liver resembled a reversible reaction reaching equilibrium in approx. 20min. After 35min, 93% of the dose was recovered with the plasma and liver as protein-bound radioactivity. Most of the asialo-transferrin associated with the liver could be displaced by asialo-orosomucoid, indicating that binding of asialo-transferrin to the galactose-specific lectin on the plasma membrane of hepatocytes was not followed by a signal for endocytosis. A range of doses, up to an average of 509.2mug of asialo-transferrin per 100g body wt., resulted in progressive increments in asialo-transferrin catabolism, as evidenced by lower dose recoveries and increased concentrations of non-protein-associated radioactivity in the liver and plasma volume. These observations indicate that binding and endocytosis of human asialo-transferrin by the rat hepatocyte are distinct phenomena. Individual asialo-transferrin molecules, although readily bound by the hepatic lectin, lack either the quantity or spacing of terminal galactose residues necessary for triggering endocytosis. Although endocytosis is induced by several asialo-transferrin molecules acting synergistically, preliminary experiments with asialo-glycopeptides and other substances have so far failed to provide further insight into the chemical basis of the signal for endocytosis.


The transport of alpha-aminoisobutyrate into Crithidia fasciculata. The transport of alpha-aminoisobutyrate into Crithidia fasciculata was characterized under aerobic and anaerobic conditions. Kinetic data for alpha-aminoisobutyrate transport were consistent with the operation of a single system of broad specificity that showed no marked dependence on Na+. Under anaerobic conditions alpha-aminoisobutyrate transport was inhibited by uncouplers such as 2,4-dinitrophenol, lipophilic cations such as methyltriphenylphosphonium ion and adenosine triphosphatase inhibitors such as dicyclohexylcarbodi-imide and NaN3. A working model in which alpha-aminoisobutyrate enters this organism by an H+-symport mechanism, the electrochemical gradient of protons being maintained by an H+-translocating adenosine triphosphatase on the cytoplasmic membrane, is proposed.


The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment. 1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The 'break' points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5'-Nucleotidase activity was stimulated by benzyl alcohol, and the 'break' point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.


Effects of maternal ethanol consumption on hepatic lipid biosynthesis in foetal and neonatal rats. Effects of prolonged maternal ethanol consumption were studied on hepatic lipid content, on the rates of fatty acid synthesis and on the activities of enzymes involved in fatty acid synthesis in the livers of foetal and suckling neonatal rats. Prolonged maternal ethanol consumption resulted in a significant increase in the contents of hepatic total lipids, triacylglycerols and plasma unesterified fatty acids in foetal and neonatal rats. Studies in vitro with 3H2O showed that maternal ethanol consumption did not result in a significant change in its rate of incorporation into lipid fractions of foetal and neonatal livers. The rates of fatty acid synthesis showed a pronounced decrease immediately after birth, compared with the foetal stage, but increased in the adult animals. On the other hand, the highest rates of lipid oxidation were observed in the neonatal stage. Maternal ethanol consumption resulted in a significant decrease in the rates of [14C]palmitate oxidation to 14CO2 by both the foetal and neonatal livers. Maternal ethanol consumption did not result in an increase in the activities of any of the lipid-synthesizing enzymes tested throughout the period of development. Although increased fatty acid synthesis does not seem to be the mechanism for the accumulation of these lipids, decreased oxidation of the lipids may be partly responsible for the lipid accumulation.


Effect of oestrogen and 1,25-dihydroxycholecalciferol on 25-hydroxycholecalciferol metabolism in primary chick kidney-cell cultures. Primary cultures of chick kidney cells convert 25-hydroxycholecalciferol into more-polar metabolites. Cells from vitamin D-deficient chicks have high 25-hydroxycholecalciferol 1 alpha-hydroxylase (1 alpha-hydroxylase) activity, but no 25-hydroxycholecalciferol 24-hydroxylase (24-hydroxylase) activity. Physiological concentrations of 1,25-dihydroxycholeclaciferol suppress 1 alpha-hydroxylase and induce 24-hydroxylase activity. The inhibition of 1 alpha-hydroxylase preceded the induction of 24-hydroxylase. In contrast, oestradiol-17 beta had no effect on the activity of either hydroxylase under a variety of experimental conditions. These results clearly demonstrate that 1,25-dihydroxycholecalciferol, but not oestrogen, acts directly on the kidney cells to regulate the metabolism of 25-hydroxycholecalciferol.


Phosphorylation of adenosine monophosphate in the mitochondrial matrix. The origin of the GTP needed for th phosphorylation of AMP in the mitochondrial matrix was investigated. When short-chain fatty acids are metabolized by hepatocytes, AMP is readily formed within the matrix by the butyryl-CoA ligase (AMP-forming) reaction (EC 6.2.1.2). The rate of matrix AMP formation in rat hepatocytes was calculated from the rate of ketone-body formation. The rate of the reconversion of matrix AMP into ADP by GTP-AMP transphosphorylase is limited by the rate of supply of GTP. GTP can be formed either by succinic thiokinase (EC 6.2.1.4) or by nucleoside diphosphokinase (EC 2.7.4.6). The rate of the succinic thiokinase reaction was calculated from turnover of the tricarboxylic acid cycle and this was calculated from the rate of O2 consumption and ketone-body formation. The results show that nucleoside diphosphokinase can make a major contribution (up to 80%) to the supply of GTP under the test conditions.


Uptake of substrates for milk-fat synthesis by lactating-rabbit mammary gland. 1. A cannulation technique is described for measuring arteriovenous differences across the lactating-rabbit mammary gland. 2. Analysis of milk obtained before and after surgery shows no effect of cannulation on milk constituents. 3. Results of blood analysis show significant net changes in the concentrations of glucose, acetate, 3-hydroxybutrate, triacylglycerols and non-esterified fatty acids across the mammary gland. 4. The molar proportions of individual fatty acids in both the triacylglycerol and non-esterified fatty acid fractions did not alter between the arterial and venous samples. 5. The extraction rates are compared with those obtained from other species.


Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient. Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.


Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation. 1. A crude lysosomal fraction obtained by differential centrifugation of a rat liver homogenate was subjected to zonal centrifugation in iso-osmotic self-generating gradients composed of modified colloidal silica (Percoll). Analysis of relevant marker-enzyme activities shows a continuous band of considerably purified lysosomal particles in the density range 1.04--1.11 g/ml. 2. A relationship between age and buoyant density of the parenchymal lysosomal subpopulations is indicated by the distribution of 125I-labelled asialoglycoproteins in the heterogeneous lysosomes during the catabolism of the glycoprotein. The labelled asialoglycoprotein first appeared in lysosomal particles of low density, which with time progressively acquired a higher density. Furthermore, 30 min after administration the 125I-labelled asialocaeruloplasmin recovered in the light lysosomes was less degraded than the material recovered in the heavy lysosomes. 3. A lysosomal enzyme (arylsulphatase) was found to possess considerably higher isoelectric points in the heavy lysosomes than in the light lysosomes, which is consistent with a relationship between age and density of the lysosomes.


Turnover of metallothioneins in rat liver. Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.


Is a urea cycle present in insects?

Is a urea cycle present in insects? 1. The presence of appreciable activity of the urea-cycle enzymes in the tissues of Sarcophaga ruficornis, a carnivorous dipteran insect, all through its life-cycle appears significant in view of their total absence barring arginase (L-arginine ureohydrolase, EC 3.5.3.1) in the phytophagous lepidopteran eri silkwork Philosamia ricini at any stage of development. Further, the variation of all these enzymes all through its development suggests the possibility of the operation of the Krebs-Henseleit urea cycle in this carnivorous insect. 2. The almost parallel behaviour of arginase and ornithine delta-transaminase (L-ornithine-2-oxo acid aminotransferase, EC 2.6.1.13) in both the insects suggests another important role of the former in proline biosynthesis in insects. 3. High proteolytic activity accompanied with significant protein depletion and simultaneous increase in arginine is suggestive of the degradation of proteins and peptides.


Diminished excretion of polyamines from BHK-21/C13 cells exposed to methylglyoxal bis(guanylhydrazone). Methylglyoxal bis(guanylhydrazone) (1,1'-[methylethanediylidine)dinitrilo]diguanidine) inhibited the growth of BHK-21/C13 cells in monolayer cultures. Accumulation of spermidine and spermine was inhibited, whereas the accumulation of putrescine was increased. The intracellular spermidine/spermine molar ratio decreased conly slightly after exposure of the cells to 20 micrometer-methylglyoxal bis(guanylhydrazone) for 1 day. Cells incubated in the presence of the drug released less polyamine into the culture medium that did control cells, the polyamine released consisting almost exclusively of spermidine, both free and as a conjugated form.


Reversal by triton WR-1339 of ethynyloestradiol-induced hepatic cholesterol esterification. RATS TREATED WITH ETHYNYLOESTRADIOL HAVE MARKED HYPOLIPIDAEMIA: serum cholesterol is decreased to 5%, triacylglycerol to 10% and phospholipid to 70% of control concentrations. Loss of serum cholesterol follows an exponential decay, with a half-life of 1.13+/-0.09 days. After 4 days of treatment, serum cholesterol concentrations remain relatively constant (ranging from 1 to 20mg/100ml) for at least 30 days. There is a concomitant 20-fold decrease in the d<1.21 fraction of serum proteins and a similar decrease in serum apolipoproteins as measured by sodium dodecyl sulphate/10%-polyacrylamide-gel electrophoresis. The activity of hepatic microsomal acyl-CoA-cholesterol O-acetyltransferase (EC 2.3.1.26) was significantly increased by ethynyloestradiol treatment (P<0.05). This activation caused hepatic cholesteryl esters containing mainly C(18:1) fatty acids to increase linearly as serum cholesterol concentrations decreased (r=0.9675, P<0.001). Triton WR-1339, a non-ionic detergent that inhibits lipoprotein catabolism, was used to estimate hepatic lipid secretion by measuring the increment in serum lipids after its administration. At 15h after Triton WR-1339 administration, serum cholesterol concentrations were increased equally in both control and ethynyloestradiol-treated rats. In contrast, the increment of serum triacylglycerol of treated rats was 40% of that found in control rats, indicating that ethynyloestradiol inhibits hepatic triacylglycerol secretion. Triton WR-1339 inhibited the oestrogen activation of hepatic microsomal acyl-CoA-cholesterol O-acyltransferase and restored hepatic cholesteryl ester concentrations to normal values. These data suggest that ethynyloestradiol and its pharmacological ;antagonist' Triton WR-1339 alter hepatic triacylglycerol secretion via a mechanism associated with changes in hepatic cholesterol esterification.


The identification of rat intestinal membrane enzymes after electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate. Brush-border membranes were isolated from the rat small intestine and then treated with sodium dodecyl sulphate under non-reducing conditions at room temperature. Analysis of the solubilized components by polyacrylamide-gel electrophoresis identified three major glycoproteins that co-migrate with glucoamylase-maltase-sucrase, lactase and isomaltase-maltase-sucrase activities. High activities of alkaline phosphatase and trehalase were detectable, but they could not be attributed to distinct co-migrating protein bands. Analysis of mucosa from the distal small intestine by the same methods showed a pattern of bands different from that obtained with the proximal intestine, which appeared to correlate with the relative deficiency of some of the enzymes in the distal region.


The lipid composition of a barley mutant lacking chlorophyll b. The acyl-lipid composition of a barley mutant that contained no detectable chlorophyll b was studied. This mutant contained chloroplasts that were much less organized than chloroplasts of normal barley. The mutant contained all the normal acyl lipids, with small increases in the relative concentration of phosphatidylglycerol and diacylsulphoquinoglycerides was unchanged, but most other lipids of the mutant barley contained lower amounts of alpha-linolenic acid compared with normal. There was no difference in the transhexadec-3-enoic acid content of phosphatidylglycerol, which was evidence against this lipid being involved in grana stacking.


Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol. 1. The influence of halogen substituents on the 1,2-dioxygenation of catechols was investigated. The results obtained with the two isoenzymes pyrocatechase I and pyrocatechase II from the haloarene-utilizing Pseudomonas sp. B 13 and the pyrocatechase from benzoate-induced cells of Alcaligenes eutrophus B.9 were compared. 2. Substituents on catechol were found to interfere with O2 binding by the two isoenzymes from Pseudomonas sp. B 13, whereas the Km value for catechol kept constant at different O2 concentrations. 3. Electron-attracting substituents decreased the Km values for catechols. 4. Results from binding studies with substituted catechols demonstrated narrow stereospecificities of pyrocatechase I from pseudomonas sp. B 13 and the pyrocatechase from alcaligenes eutrophus B.9. In contrast, a low steric hindrance by substituents in the binding of catechols with pyrocatechase II was observed. 5. Low pK'1 values of substituted catechols resulted in low Michaelis constants. 6. Electron-attracting substituents such as halogen decreased the reaction rates of catechol 1,2-dioxygenation. The correlation of the Vmax. values observed with pyrocatechase II from Pseudomonas sp. B 13 with the substituent constant sigma+ (Okamoto--Brown equation) was distinctly greater than with Hammett's sigma values. The corresponding logVmax. against sigma+ correlation for pyrocatechase I was considerably disturbed by steric influences of the substituents.


Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad. 1. Two catechol 1,2-dioxygenases, pyrocatechase I and pyrocatechase II, were found in 3-chlorobenzoate-grown cells of Pseudomonas sp. B 13. The latter enzyme showed high relative activities with 3- and 4-chlorocatechol compared with catechol. 2. In benzoate-grown cells, only pyrocatechase I was induced. It was purified 29-fold with a final specific activity of 20 mumol of catechol oxygenated/min per mg of protein and an overall yield of 22%. Because of the instability of pyrocatechase II on chromatography and dialysis, no increase of specific activity was obtained during the purification experiments. 3. Molecular weights of pyrocatechase I and pyrocatechase II were 82000 and 67000 respectively. 4. For both pyrocatechases the pH optimum was found to be at 8.0.5. Inhibitions of the two pyrocatechases by Cu2+ and Hg2+ ions and p-chloromercuribenzoate were different. The effect on pyrocatechase I after incubation for 20 h with the heavy metals was decreased by addition of 1 mM-2-mercaptoethanol to the reaction mixture. The inhibition of pyrocatechase II was even enhanced under these conditions. 6. Extradiol cleavage of 3-methylcatechol in addition to intradiol fission at a ratio of 1:14 was observed only with pyrocatechase I.


Mechanisms of replenishment of nuclear androgen receptor in rat ventral prostate. 1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2mug of [(3)H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [(3)H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [(3)H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5-60min interval after the administration of [(3)H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.


Effect of non-histone chromosomal proteins on transcription in vitro in sea-urchin. Non-histone chromosomal proteins prepared from chromosomal material of the sea-urchin Paracentrotus lividus affect RNA synthesis in vitro. 1. The extent of transcription can be radically changed from inhibition to stimulation, depending on the DNA/non-histone chromosomal proteins ratio. 2. A correlation exists between stage of development and influence on transcription. 3. Non-histone chromosomal proteins exert their action by intervening directly on some initiation step of RNA synthesis, as shown by the numbers of initiation events that take place in their presence or absence. 4. Stimulatory activity is observed only in restrictive conditions of ionic strength and temperature. These observations are in agreement with models that predict for non-histone chromosomal proteins a regulatory role on the transcription process exerted through a modulation of promoter availability.


Boundary lubricating ability of synovial fluid in degenerative joint disease. The boundary lubricating ability of eleven synovial fluids was measured in a miniaturized latex--glass test system. The specimens were obtained at necropsy from knees in which the degree of degenerative joint disease varied from none to very severe. The lubricating ability of the fluid was independent of the viscosity over a wide range of shear rates. It was not diminished even in advanced lesions. In two additional fluids, the mucin clot was poor; the lubricating ability of one of these was compromised. Thus, although degenerative joint disease, during its quiescent stages, is not associated with defective synovial lubrication, the possibility that transient defects might lead to cartilage wear during life has not been excluded. The measurements are believed to be valid indicators of boundary lubricating ability under physiological conditions despite the fact that the test surfaces were not cartilaginous and the loading was relatively low (up to 47 pounds per square inch).


Differential effects of propranolol on lymphocyte rosette formation and response to plant mitogens. The effects of propranolol on various lymphocyte functions were studied to gain a better understanding of the recently demonstrated suppressive effect of propranolol on rheumatoid factor production. D- and L-propranolol at a concentration of 1 X 10(-4)M inhibited the formation of human EA rosettes. The inhibition occurred within one minute of adding the compounds, was reversible, and did not affect cell viability. Addtion of propranolol to preformed EA rosettes failed to disaggregate them. Patching and capping of SIg by an Fab'2 anti-IgG were inhibited at 2.5 X 10(-5)M and above. Propranolol at 2.5 X 10(-5)M also inhibited lymphocyte response to phytohemagglutinin and pokeweed mitogen without evidence of cell toxicity by trypan blue staining or absolute numbers of surviving cells. Congeners of propranolol with mainly beta adrenergic blocking properties did not show inhibitory effects. The inhibitory activities of propranolol are interpreted in terms of propranolol's membrane stablizing effects and ability to interfere with membrane receptor movement.


Synovial fluid lactic acid. A diagnostic aid in septic arthritis. Lactic acid concentrations in the synovial fluid of 84 patients with acute monoarticular arthritis were determined by gas liquid chromatography. Lactic acid values in 27 cases of nongonococcal septic arthritis were strikingly higher (mean 1170 mg/100ml) than in 45 cases of inflammatory or degenerative arthritis (mean 34 mg/100 ml), as well as in 12 cases of gonococcal arthritis (mean 27 mg/100 ml). With the proper equipment, determination of lactic acid can be a relatively rapid, reliable procedure. Synovial fluid lactic acid concentrations therefore can be used as a rapid, supplemental diagnostic aid in differentiating nongonococcal septic arthritis from both gonococcal and nonseptic acute arthritis.


Temporal arteriography. Analysis of 21 cases and a review of the literature. To define the role of temporal arteriography in the diagnosis of giant-cell arteritis, the results of temporal arteriography in 21 patients who had temporal artery biopsies were reviewed. Abnormal angiography was found in all patients with biopsy-proven giant-cell arteritis, in some patients with polymyalgia rheumatica and normal biopsy, and in some patients with neither polymyalgia rheumatica nor giant-cell arteritis. No pattern of angiographic abnormality typical for giant-cell arteritis could be found. Results suggest temporal arteriography is a sensitive but nonspecific procedure in the diagnosis of giant-cell arteritis and must be coupled with biopsy of angiographic abnormalities for confirmation of diagnosis.


Herpes zoster in patients with systemic lupus erythematosus. In the context of prospective trials of immunosuppressive drugs in systemic lupus erythematosus (SLE) nephritis, 83 patients were studied with regard to development of herpes zoster. Herpes zoster was found to occur with high frequency (21%) in patients with SLE nephritis treated with immunosuppressive agents. The course of herpes zoster was benign: no deaths occurred and only 2 of the 18 patients developed generalized disease, which resolved without sequelae. Specific antiviral therapy was not necessary and there appears to be no need to decrease immunosuppressive medications. Zoster occurred when the SLE was relatively inactive and did not exacerbate the SLE. No statistical difference in the incidence of zoster was found among the patient groups treated with different immunosuppressive regimens.


Effect of inherited deficiency of the fifth component of complement on arthritis induced in mice by Mycoplasma pulmonis. Mycoplasma pulmonis inoculated parenterally into mice deficient in the fifth component of complement (C5) caused a chronic arthritis of significantly greater magnitude than in immunologically normal mice. During the chronic phase of arthritis M pulmonis organisms were isolated from the joints and organs of C5 deficient mice more frequently and in larger numbers than from immunologically normal mice. The implications of the results are discussed in relation to the pathogenesis of M pulmonis induced arthritis and human connective tissue diseases.


Regional migratory osteoporosis. A denervation disease. Regional migratory osteoporosis is an uncommon entity charcterized by sudden attacks of migrating lower extremity paraarticular pain, local edema, and muscle atrophy; the disease is verified by radiologic demineralization and bone scan uptake. The etiology and pathogenesis are unknown. We describe a case of regional migratory osteoporosis followed over nine years by serial electromyographic studies documenting denervation patterns coincident in time and location of each acute attack. The substantiation of denervation in regional migratory osteoporosis is of both diagnostic and pathogenetic significance.


Central retinal vein occlusion complicating systemic lupus erythematosus. Retinal lesions occur in 25 to 30% of patients with systemic lupus erythematosus (SLE). Visual loss, however, is rare. A patient who developed central retinal vein occlusion and blindness during the course of active SLE is described. The pertinent literature is reviewed with attention to possible pathophysiologic mechanisms.


Hemochromatotic arthropathy mimicking rheumatoid arthritis. A case with subcutaneous nodules, tenosynovitis, and bursitis. A 63-year-old man developed symmetrical polyarthritis, subcutaneous nodules at the elbows, olecranon bursitis, and recurrent tenosynovitis. He was later discovered to have idiopathic hemochromatosis. Staining of the subcutaneous nodule revealed iron deposits. These manifestations which are common to rheumatoid arthritis may be seen in hemochromatotic arthropathy.


Immunologic sex differences and the female predominance in systemic lupus erythematosus. The female predominance in many rheumatic disorders, most notably systemic lupus erythematosus, suggests that immunologic sex differences may modify susceptibility to disease. Clinical and experimental data indicate heightened humoral immunity and depressed cellular immunity in females compared with males. These differences appear to be mediated by sex hormones.


Electrocorticographic effects of topically applied scopolamine. The electrocorticographic effects of topically applied scopolamine were investigated in unanesthetized cats with high cervical transection. After subpial injection of 30 cumu scopolamine in concentrations of 10 to 20%, large amplitude intermittent sharp waves appeared in the electrocorticogram, which developed into long-lasting paroxysmal activity. This effect was antagonized by intravenous eserine, 0.1 mg/kg, when it was produced by topical scopolamine in low dosage. The acoustically evoked cortical response and the generalized epileptiform activity produced by topical succinylcholine disappeared after topical scopolamine in low dosage. It is suggested that the seizure-suppressing effect of scopolamine may be due to its cholinolytic action. The convulsive activity of topical scopolamine in high concentrations may be accounted for by its depolarizing, synchronizing, disinhibiting, and acetylcholine-releasing effects.


[Scintigraphic changes on the liver and spleen in urticaria pigmentosa]. In 36 patients with mastocytosis pathological changes in liver and spleen were scintigraphically demonstrated. These changes are due to the histologically proven involvement of the reticuloendothelial system. The infiltration of the liver by mast cells was not evident in liver biopsy.


[Syntopy of Bowen's disease and Lewisite scar]. Description of a case of Bown's disease in an area of the right lower leg after lewisite contamination 30 years ago. The possible influence of lost derivatives on carcinogenesis is discussed.


[Psoriasis pustulosa: iron as a therapeutic agent?]. In a case of psoriasis pustulosa the benefit of iron-therapy is discussed, as it is described in literature. Diagnostic criteria are stated that are necessary for the diagnosis: iron deficiency, also in psoriasis pustulosa. Only true iron deficiency justifies iron medication. Even in its pustular variants per se psoriasis is no indication for high-dose parenteral iron-application.


Catecholamine sensitivity in hyperthyroidism and hypothyroidism. 1. Catecholamine sensitivity in hyper- and hypothyroidism has been studied using a standardised isoprenaline sensitivity test. 2. Seven patients with hyperthyroidism and seven with hypothyroidism were tested both when showing evidence of thyroid dysfunction and again when euthyroid. 3. No significant differences were seen in heart rate responses to isoprenaline when patients became euthyroid compared to their response when either hyperthyroid or hypothyroid. 4. These results indicate that sensitivity to catecholamines is not altered in thyroid dysfunction and, in particular, that hypersensitivity does not occur in spontaneous hyperthyroidism.


Iotroxamide studies in man--plasma binding, renal and biliary excretion studies in jaundiced and anicteric patients. 1.A decrease in the plasma binding of iotroxamide was found in jaundiced people. 2. The decrease in binding was associated with an enhancement in renal excretion. 3. Iotroxamide consistently gave higher biliary iodine concentrations than ioglycamide.


The effects of low doses of diazepam on human performance in group administered tasks. 1. The effects of diazepam in 5 mg dosage were assessed on a range of psychological tasks. Seventy-eight healthy subjects were tested in an independent groups design; subjects were randomly assigned to either control, placebo or drug group. Treatments were administered orally under double blind conditions. 2. Auditory vigilance performance was unimpaired, in terms of (a) correct detections, (b) false alarms or (c) the subjects' estimates of the duration of the task. 3. The short term retention of digit strings was impaired by diazepam (P less than 0.05), especially for those digits presented in the middle of the sequence. 4. Searching for a few letters among many was significantly impaired by diazepam (P less than 0.01). 5. Diaepam had no effect on performance at a mental arithmetic task; neither was there a placebo effect. 6. Results were discussed in the light of the characteristics of drug sensitive tasks. It was concluded that characteristics such as feedback of results, monotony, and memory load are more likely to be drug sensitive when in combination than in isolation.


Digoxin compliance in patients from general practice. 1. Compliance with digoxin therapy has been assessed in a group of fifty patients receiving the drug in general practice but not attending hospital. 2. Compliance was estimated by comparing plasma digoxin concentrations before and after a 10 day period of measured digoxin consumption and by tablet counting. 3. Twelve patients had plasma concentrations which increased by more than 0.4 ng/ml during monitored intake and eight other patients took less than 80% of their tablets. These twenty patients were considered non-compliant. 4. A further three patients in whom plasma digoxin levels were zero when first seen but increased substantially during the run-in period were also adjudged non-compliant. 5. Non-compliance with prescribed digoxin dosage occurred, therefore, in 46% of the patients studied.


Direct measurement of propranolol bioavailability during accumulation to steady-state. 1. A high performance liquid chromatographic method for the determination of propranolol in human plasma and blood has been developed and used to confirm that cumulation occurred during chronic oral administration, steady-state being achieved within 48 h of beginning 80 mg of the drug every 8 h. 2. The method was adapted to measure [H3]-propranolol and native drug in the same blood sample and was applied to determine simultaneously the disposition of i.v. ([H3]-propranolol) and orally (non-labelled) administered drug after single oral dose of 80 mg and when steady-state had been established on an 80 mg, 8-hourly regimen. 3. Using this approach it was possible to show that a reduced oral clearance at steady-state was associated with a smaller reduction in systemic (i.v.) clearance and no change in liver blood flow. A direct estimate of bioavailability was also possible and was found to be increased at steady-state compared with a single oral dose. 4. We conclude that the accumulation of propranolol during the attainment of steady-state is due to a reduction in intrinsic clearance, resulting in reduced presystemic hepatic extraction.


Analysis of synergy between cyclophosphamide therapy and immunity against a mouse tumour. C3H/He and CBA/T6T6 mice which share the H2(k) haplotype were compared for their capacity to survive challenges with the C3H-derived fibrosarcoma BP8. It was found:(1) The tumour grows at the same rate with the same median survival time in matched groups of non-immunized mice from both strains after i.p. injection of tumour cells.(2) Cyclophosphamide (Cyclo) at 10 mg/kg will cure CBA mice which have received i.p. injections of 10(7) BP8, but this dose, and more intensive treatment with this drug, fails to cure C3H mice.(3) Injecting (125)IUdR-labelled tumour cells and counting (125)I loss by whole-mouse counting shows that the cytotoxic effect of Cyclo against BP8 is similar in the 2 mouse strains.(4) Cyclo itself does not cure CBA mice, for viable tumour cells are recoverable from the peritoneal cavity 10 days after CBA mice have received 10(7) BP8 followed by 10 mg/kg Cyclo.(5) CBA mice cured of BP8 ascites by Cyclo treatment will reject further i.p. inocula of BP8.(6) The strength of immunity induced by irradiated BP8 cells was directly related to the length of exposure to this antigen. An important aspect of Cyclo treatment is that it prolongs the period during which immunity may develop.(7) Immunization of CBA mice with heavily irradiated BP8, with or without Cyclo, failed to show that Cyclo depressed the capacity of CBA mice to develop cytotoxic immunity. There was some indication that animals immunized with irradiated cells plus drug did better than those with irradiated cells alone.(8) A single injection of irradiated BP8 cells into CBA mice induced weak cytotoxic immunity, as assessed by destruction of a subsequent challenge with BP8, but these mice died from tumour more rapidly than non-immunized controls. It is suggested from these data that immunological enhancement may not always be due to blocking of cytotoxic immunity.


Failure of preoperative C. parvum vaccine to modify secondary disease following excision of two non-immunogenic murine carcinomas. Sadler and Castro (1976) reported that a single dose of C. parvum vaccine given i.p. or i.v. to mice 4 days before excision of subcutaneous transplants of Lewis lung carcinoma significantly reduced the incidence of lung metastases in the operated mice. In similarly designed experiments, using 2 different carcinomas of spontaneous origin in our own inbred mouse colonies, we were unable to demonstrate any influence of C. parvum vaccine on the incidence or latent period of secondary disease in operated mice. We discuss possible reasons for our failure to reproduce the findings of Sadler and Castro.


Biphasic change of proton magnetic relaxation times during azo-dye hepatocarcinogenesis. For the first time, change in the proton longitudinal relaxation times (T1) of rat tissues has been examined throughout the whole process of azo-dye hepatocarcinogenesis. Two maxima of the T1 values were observed for liver, on Day 60 and after Day 120, and these changes correlated well with the changes in water content. The first peak was ascribed to the immature hepatocytes of hyperplastic nodules, and the second peak to the developed hepatoma cells. The significance of the change in T1 values as a preneoplastic change is discussed.


Thyroid tumours in rats and hepatomas in mice after griseofulvin treatment. Griseofulvin, an antibiotic used to treat dermatophystosis, was tested for carcinogenicity in mice, rats and hamsters. Three groups of mice and rats were given the drug in powdered diet in alternating 5-week periods for life, at dose levels of 3.0%, 1.5% and 0.3% (mice) and 2.0%, 1.0% and 0.2% (rats). A group of mice and 3 groups of hamsters received continuous daily treatment for life with griseofulvin at 3.0%, 1.5%, 0.3% and 0.1% dose levels respectively. A significant incidence of hepatic tumours was observed at the 2 higher treatment levels in mice. Also, statistically significant rates (P less than or equal to 0.001 and/or P less than or equal to 0.020) of thyroid tumours, indicating a dose-response, were recorded in male rats at the 2.0%, 1.0%, and 0.2% dose levels, and in females at the 2.0% and 1.0% dose levels. Hamsters did not develop neoplasms in response to treatment at any level.


Effect of glycerol on local and systemic carcinogenicity of topically applied tobacco condensate. When glycerol was added to tobacco smoke condensate in acetone solvent, the topical carcinogenicity and the ability to produce epithelial hyperplasia in mice was reduced. Two doses of condensate were applied, combined with 2 concentrations of added glycerol. Age-standardized results show that glycerol reduced the incidence of tumours and malignant tumours and of hyperplasia in animals not developing skin tumours. The relative incidences of malignant tumours, benign tumours, hyperplasia and unaffected skin suggest that there is a sequential relationship (i.e. normal skin to hyperplasia to benign neoplasia to malignant neoplasia) which is impeded by glycerol. There was no systemic effect attributable to the condensate.


Effects of insulin, human placental lactogen and human growth hormone of DNA synthesis in organ cultures of benign human breast tumours. Nineteen benign human breast tumours, from 19 premenopausal patients, were processed into slices and each tumour was individually cultured for 2 days in Medium 199. The effects of bovine insulin (5.0 microgram/ml) human placental lactogen (10.0 microgram/ml) and human growth hormone (10.0 microgram/ml) on 3H-thymidine incorporation into DNA were determined on the cultured tumour slices. Insulin and human placental lactogen significantly (P less than 0.01) increased the mean incorporation of [3H]TdR into DNA, whereas human growth hormone was ineffective. These results provide evidence that insulin and human placental lactogen, but not human growth hormone, may be important factors in the aetiology of benign human breast tumours.


Growth of human gliomas in immune-deficient mice: a possible model for pre-clinical therapy studies. Thirteen gliomas from 55 neurosurgical specimens, derived from 25 adults and 30 children, have been successfully grown as subcutaneous xenografts in immune-deprived or nude mice. Only 2 of the 30 paediatric specimens implanted (6.7%), a medulloblastoma and an astrocytoma Grade III, have grown compared with 11 of the 25 adult specimen (44%) which were mostly astrocytomas Grade III. Tumour growth usually occurred several months after implantation, and karyotypic analysis confirmed their human origin in all cases. The histopathology of xenografted tumours correlated with the original surgical material, both after initial implantation and when tumours had been passaged several times. Observations on tumour growth in various types of immune-deprived mice indicated that, within certain limits, the immunological competence of the host mouse did not relate to take rates of primary implants, but could affect the take rate of passaged tumours.


Neoplasia and dysplasia of the cervix uteri and contraception: a possible protective effect of the diaphragm. Among the 17,032 women included in the Oxford-Family Planning Association contraceptive study, 65 developed biopsy proven cervical neoplasia (including dysplasia) prior to 1 September 1977. The incidence rate in diaphragm users (0.17 per 1000 woman-years of observation) was much lower than the rates in oral contraceptive users or intrauterine device users (0.95 and 0.87 respectively). This difference could not be explained in terms of confounding variables, nor was it attributable to a lower frequency of cervical smearing among diaphragm users within the clinics. Detailed information about age at first intercourse, numbers of sexual partners and the frequency of cervical smearing outside the clinics was obtained from 52 of the women with cervical neoplasia and 139 matched controls. Diaphragm users were less likely to have had coitus at an early age and had had materially fewer sexual partners than users of the other two methods of contraception. After adjusting for the effects of these variables, however, the risk of cervical neoplasia in diaphragm users was still only about one quarter that in the users of the other methods. Patterns of smearing varied little between users of the various contraceptive methods. Smoking emerged as a major "risk factor" for cervical neoplasia in this study. This probably implies that the smoking habit reflects some important aspect of sexual behaviour relevant to the production of the disease that we have been unable to measure.


Serum ribonuclease activity in cancer patients. A study was made of the levels of ribonuclease (RNase) in human serum, using 2 independently collected banks of samples from Scripps Clinic and Research Foundation and the Mayo Clinic, each bank representing more than 100 individuals. These serum samples originated from a cross-section of normal individuals, smokers, patients with benign tumours, and patients with a variety of neoplasms. Elevated levels of serum RNase occurred in 68% of the samples from individuals with malignant disease. Elevated levels also occurred in 24% of the samples from individuals with benign tumours and in 38% of the smoker controls from the Mayo Clinic serum bank. Using ion-exchange chromatography, pooled sera from normal individuals and cancer patients were fractionated by differential salt elution. Each pool showed 2 distinct peaks of RNase activity, and both peaks were elevated to the same degree in the cancer serum pools. Similar results were obtained after thin-layer-gel isoelectric focusing of both normal and cancer sera; no new species of RNase could be detected in the sera of patients with malignant diseases. The results suggested a generalized nonspecific increase in serum RNase in these patients.


Mechanism of impaired glucose tolerance in patients with neoplasia. The disappearance rate (k) of i.v. glucose was measured in cachectic and non-cachectic cancer patients and tumour-free controls. The respective k values were found to be 1.06 +/- 0.27 (mean +/- s.d.), 1.64 +/- 0.34 and 1.63 +/- 0.23. Of the other parameters measured, only plasma albumin level was found to vary significantly amongst the 3 categories, the mean level being the lowest in cachectic cancer patients. The means of total plasma protein, fasting blood glucose and plasma liver enzyme concentrations were similar in the 3 groups. Glucagon, a potent insulin secretogogue, failed to augment the fasting insulin level in cachectic but did so in non-cachectic cancer patients. Taken together, the findings suggest that the reduced glucose tolerance in patients with neoplasia is due to impairment of insulin release exhibited predominantly by ill-nourished advanced cancer patients having a moderate to sever degree of hypoalbuminemia.


Lodgement and extravasation of tumour cells in blood-borne metastasis: an electron microscope study. Soon after i.v. injection of ascites hepatoma cells of rat, 3 types of tumour-cell emboli were found in arterioles and capillaries of the lung. The first type had marked aggregation of platelets and deposition of fibrin. Many were seen when tumour cells with high thromboplastic activity (AH 130) were injected, and were often followed by detachment and fragmentation of endothelial cells. The second type had loosely aggregated platelets and the third type had no aggregation of platelets or deposition of fibrin. The latter 2 types were mainly seen when the tumour cells with low thromboplastic activity [AH 130 F(N)] were injected, and they did not accompany severe structural changes of the endothelial cells. Tumour cell-platelet complexes appeared to be induced by tissue thromboplastin released from tumour cells rather than from the endothelial cells. One to 6 h after injection of AH 130, tumour cells were found beneath the endothelial cells detached from the basement membrane in areas with microthrombi. Breaching of the endothelial cells with the processes of tumour cells was also seen then. Intrusion of the processes of tumour cells into the endothelial cells was noted in groups injected with either AH 130 or AH 130 F(N), but not in the junctions of the endothelial cells. Metastatic foci 3 days after the injection of AH 130 were more frequent than in the rats injected with AH 130 F(N). These results indicate that thromboplastic activity of tumour cells might be important in forming microthrombi in the lodgement phase and might be one of the factors facilitating blood-borne metastasis.


Tumour-necrosis factor from the rabbit. I. Mode of action, specificity and physicochemical properties. Sera from rabbits injected with BCG and then with endotoxin contain a factor (tumour-necrosis factor TNF) which, even at high dilutions, is cytotoxic in vitro for mouse L cells and some other cell lines. Using a 51Cr-release assay, cytotoxicity was detected as early as 7-8 h after addition of TNF serum to L cells and cell death was evident microscopically by 24 h. TNF was cytotoxic at 37 degrees C but not at 21 degrees C or 4 degrees C, and acted on both dividing and non-dividing cells. The antimetabolites sodium azide and dinitrophenol partially protected L cells from TNF, suggesting that actively metabolizing cells are the most sensitive. Treatment of L cells with trypsin did not delay cytotoxicity nor was cytotoxicity inhibited in the presence of various saccharide derivatives of cell-surface glycoproteins. Rabbit TNF was remarkably stable with a mol. wt. of 40-50,000. It was eluted with the more acidic serum proteins on ion-exchange chromatography, but precipitated in 50%-saturated ammonium sulphate. Sensitivity to TNF could not be correlated with tumourigenicity of several animal and human lines tested nor with the production of C-type viruses.


Tumour-necrosis factor from the rabbit. II. Production by monocytes. Mononuclear cells from normal rabbit blood were cytotoxic to a number of cell lines in vitro. The cytotoxic cells were contained in the monocyte-enriched fraction adherent to plastic. Supernatants of the monocyte-enriched fraction had the same cytotoxic specificity as the parent cells. The cytotoxic factor precipitated in 50% saturated ammonium sulphate solution and on gel filtration was heterogeneous with a mol.-wt. range of 30--50,000 u. On the basis of specificity and molecular characteristics, this cytotoxic factor closely resembles rabbit tumour-necrosis factor (TNF) suggesting that TNF or a closely related factor is a normal product of mononuclear phagocytes.


Effect of phenobarbital on plasma levels of cyclophosphamide and its metabolites in the mouse. We have studied the quantitative pharmacokinetic differences of individual metabolites and unchanged cyclophosphamide (CPA) in control and phenobarbital-treated animals, using radiolabelled CPA together with thin-layer chromatography. On Day 0, one group was started on phenobarbital drinking water and one group stayed on regular acid water. P388 leukaemia, (10(6) cells i.p.) was administered to all mice on Day 8, and 2 days later both groups of mice were given i.p. CPA (200 mg/kg) with 14C-CPA (0.2 muCi per mouse). At 5--60 min after CPA administration, groups of 10 mice were killed and their blood collected for assay of parent compound and metabolites in plasma. Phenobarbital pretreatment reduced CPA and phosphoramide mustard CXT (concentration x time) by 66+% and 27+%, respectively. Assuming that phosphoramide mustard is both the ultimate cytotoxic form of CPA and the blood-transport form, the reduction of CPA by phenobarbital would predict a decreased therapeutic effect. The assay methods in this study will be used in the future to determine the importance of this potential drug interaction in man.


Metal chelates as anti-cancer agents. II cytotoxic action of palladium and platinum complexes of 6-mercaptopurine and thioguanine. The metal complexes Pd(MP)2.2H2O, Pt(MP)2H2O (MPH=6-mercaptopurine), Pt(AMP2.3H2O and Pd3(AMP)4Cl2(AMPH).4H2O (AMPH=thioguanine) have been isolated. They were screened for anti-tumour activity in the L-1210 lymphoid leukaemia test system in mice. All 4 show marked anti-tumour activity, the complex Pt(AMP)2.3H2O giving a T/C of 185 at the optimum dosage. However, the anti-tumour activity of the metal complexes is somewhat less than that shown by the parent purines under the same conditions.


High-altitude acclimatization and proteinuria in East Africa. Seven climbers spent 6 weeks in the East African mountains, at heights of up to 5890 m. The mean protein urine concentration in morning specimens was over 100 mg/100 ml after climbs during the first 12 days but fell to 15 mg/100 ml during subsequent climbs. The highest concentrations (100-300 mg/100 ml) were in 5 climbers with peripheral oedema, 3 with obvious high-altitude illness. The findings implicate the kidney in high altitude acclimatization and illness.


Aspiration needle biopsy for solitary, peripheral lung lesions: a review of 50 examinations. Percutaneous aspiration needle biopsy forms a simple method of obtaining histological material from solitary peripheral lung lesions. A new method for separating and preparing the material for histological examination is described. The results in 50 cases are recorded with a positive diagnosis in 46. The number of symptomatic complications with this technique is low. The clinical place of the biopsy in management is discussed.


The association of adenocarcinoma of the lung and blastomycosis from an unusual geographical location. A case of North American blastomycosis acquired outside the American continent and presenting with a left hilar mass is reported. The progressive hilar enlargement, despite adequate antifungal treatment, raised the possibility of drug resistance, concomitant tuberculosis and carcinoma. Non-invasive diagnostic studies including cultures, cytology and fibreoptic bronchoscopy were negative. On thoracotomy, both blastomy cosis and a poorly undifferentiated adenocarcinoma were evident.


The pathogenesis of miliaria rubra. Role of the resident microflora. Anhidrosis was induced in volunteers by covering the skin with an impermeable plastic film. The degree of sweat suppression and miliaria after a thermal stimulus was directly proportional to the increase in the density of resident aerobic bacteria, notably cocci. No anhidrosis resulted when antibacterial substances were used to prevent the expansion of the microflora. Histological study showed a PAS-positive diastase resistant amorphous mass deep within the acrosyringium after 2 days of occlusion, accompanied by a periductal dermal infiltration of leukocytes. After another 2 days the duct became clogged by an amalgam of degenerating leukocytes. This impaction sloughed after about 3 weeks as a result of epidermal renewal. Colonies of bacteria were never found within the ducts. It is postulated that cocci secrete a toxin which injures luminal cells and precipitates a cast within the lumen. Infiltration by leukocytes creates an impaction which completely obstructs the passage of sweat for several weeks.


Epidermal differentiation in normal and growth-retarded infants: studies in two animal models and in human babies. The influence of prenatal growth retardation on epidermal growth and keratinization was studied in small-for-dates human babies, runt piglets and in rat fetuses subject to maternal protein deprivation. In the human babies and rat fetuses growth retardation was associated with reduced epidermal growth but normal patterns of differentiation were present. Thus, epidermal thickness was less and keratinizing zones narrower than seen in normal weight individuals. In the runt (small-for-dates) piglet, epidermal development differed from that seen in normal weight piglets of the same gestational age and from that seen in the human and rat. The epidermis was thicker with a negligible stratum granulosum and with the stratum corneum containing nucleated cells, a condition resembling 'parakeratosis'. This pattern was less often seen in normal weight piglets. Differences between the skins of the three speecies studied at a perinatal stage may be related to clear dissimilarities which exist in the tissues later. Parakeratosis as a transitory phase in the development of pig skin is probably related to a higher rate of epidermal keratinization in this species than occurs in either human babies or rat fetuses.