Performance of high frequency impaired listeners with conventional and extended high frequency amplification.

This study sought to determine if a high-pass hearing aid can provide increased improvement in word recognition and consonant discrimination over that of a conventional high frequency emphasis hearing aid in listeners with hearing loss limited to frequencies above 1 000 Hz. Word and consonant discrimination were assessed in quiet and in the presence of 12 talker speech babble for 10 subjects under three listening conditions: (1) unaided; (2) wearing a conventional high frequency emphasis hearing aid, and (3) wearing an experimental high-pass instrument. The speech testing materials included: (1) Northwestern University Auditory Test No. 6; (2) California Consonant Test, and (3) eight voiceless English consonants. Results suggested that both instruments provided similar benefit in quiet for improving word recognition and resolving consonant errors. For the noise condition, however, the experimental high-pass aid provided a considerable advantage in both word recognition and consonant identification and was particularly sensitive to reducing within- and between-manner voiceless consonant confusions. Furthermore, measurements of real-ear gain revealed that the high-pass aid afforded considerably greater acoustic gain above 4 000 Hz than that shown for the conventional high frequency emphasis hearing aid,20


Auditory nerve and brain stem volume-conducted potentials evoked by pure-tone pips in the CBA/J laboratory mouse.

The short latency (less then 10 ms) auditory nerve and brain stem evoked potentials of Jewett were examined in the inbred CBA/J laboratory mouse. Tone pips, at nine frequencies from 4 to 64 kHz, were used to determine auditory nerve evoked potential (P1) thresholds. Maximum sensitivity occurred at 32 kHz, and a second sensitive area was seen at 56 kHz. With the exception of 32 kHz, as stimulus frequencies were increased in octave intervals, the 90 dB P1 amplitude also increased. P1 latency decreased as the stimulus frequency was increased up to 32 kHz, but this trend reversed with further increases of frequency. However, the P1 amplitude-latency function produced a better agreement of latency and frequency with the travelling wave theory for amplitudes less than 1 micro V, while amplitudes from 2 to 8 micro V were independent of frequency. Latency and amplitude input-output functions for responses from the four auditory brain stem loci suggest that auditory information is differentially processed as a function of frequency at each level, This is especially true for the inferior colliculus, which is most sensitive to 8-KHz stimuli.


Leptospira interrogans serovar hardjo infection of cattle.

A survey of normal cattle in the Southern Victorian statistical divisions revealed that microscopic agglutination titres to L. hardjo occur at high frequency and are distributed throughout the cattle population. These titres are difficult to interpret as they may represent recent or old exposure, with or without disease. L. hardjo infection of dairy cattle was studied in 4 herds using the microscopic agglutination and complement-fixation tests. Statistical comparisons of individual titres obtained indicated that the sensitivity of the complement-fixation test was satisfactory for diagnostic purposes, but the test was unable to differentiate between current or past infections.


Immunity in cattle to Babesia bovis after single infections with parasites of various origin.

Sixty calves, 3 to 6 months old, were vaccinated once against Babesia bovis in groups of 10, by the following methods: (a) tick infestation; (b) inoculation of virulent parasites obtained from the tick-infested animals immediately after infection; (c) inoculation of the parasites used in (b) attenuated by passage through splenectomised calves; (d) inoculation of commercially-available, living, attenuated vaccine; (e) inoculation of virulent parasites obtained from the tick-infested animals in (a) one year after infection; (f) inoculation of the parasites used in (e) attenuated by passage. All vaccinated animals were maintained tick-free and were strongly immune to challenge with a heterologous strain of B. bovis approximately 4 years after vaccination. There was no difference in immunogenicity between any of the B. bovis populations.


Salmonella contamination of meat and bone meal.

The production of meat and bone meal from 8 rendering plants was examined for the presence of salmonellas. Of 164 samples of final product 114 (69.5%) were contaminated with salmonellas. Of 65 samples, collected at various points from the production line 35 (53.8%), and of 95 samples collected from the processing environment 79 (83.1%) were found to be contaminated with salmonellas. A total of 41 serotypes were found with S. havana, S. eimsbuettel, S. ohio and S. singapore being the most frequently isolated. Pre-enrichment of 25g samples in buffered peptone water, followed by enrichment in mannitol selenite cystine broth at 42 degrees C and Muller-Kauffmann Tetrathionate Broth at 37 degrees C and plating on Bismuth Sulphite Agar was found to yield 98.5% of the salmonella-positive samples.


Observations on Salmonella infections of birds.

During the period 1961 to 1976, 29 species of Salmonella other than Salmonella pullorum were isolated from 180 accessions of birds examined at the Animal Research Institute, Yeerongpilly. These birds were submitted to the laboratory from flocks with disease or production problems. S. typhimurium was the most frequently isolated serotype being obtained from 63% of accessions. Outbreaks of systemic salmonellosis occurred most frequently in young birds and although pathological changes were most commonly observed in visceral organs they were also seen in eyes, joints and the brain. Diseases other than salmonellosis were identified in many accessions of birds with systemic or enteric salmonella infections.


Masculinisation of the external genitalia in ewes with clover disease.

Prolonged exposure of ewes to oestrogenic pasture resulted in partial fusion of the labia of the vulva and hypertrophy of the clitoris. Partial fusion of the labia was also seen in ewes which had grazed oestrogenic pastures on normal commercial properties, and was related to the amount of histological change in the cervix. This lesion may be useful as a screening test for previous exposure to oestrogenic pasture.


A degrading proteinase test to distinguish benign and virulent ovine isolates of Bacteroides nodosus.

Bacteroides nodosus was recovered from naturally occurring cases of virulent ovine footrot (VFR) and benign footrot (BFR) using an artificial culture medium which incorporated trypticase, arginine, serine and 5% agar. A degrading proteinase (DP) test was developed which measured the proteinase activity of broth cultures of B. nodosus for a period of time after organism death to assess the stability of the enzyme. The spectrophotometric measurement of the release of dye from a hide powder--azure conjugate by the action of proteinase provided an objective analysis of enzyme activity. The DP test differentiated VFR and BFR isolates and promises to be a useful laboratory method for the diagnosis of benign and virulent footrot in sheep.


Internal fixation of fractures of the third phalanx in three horses.

The technique of internal fixation of intra-articular fractures of the third phalanx using lag screw interfragmentary compression is described. Three cases are discussed and the successful treatment of infection and osteomyelitis described in one of them. Despite this potential complication internal fixation offers a better prognosis and also a more rapid return to work than non-operative treatments.


The correction of uterine torsion in a mare by caesarean section.

A 9-year-old mare exhibiting signs of colic late in pregnancy was presented. Uterine torsion was diagnosed and corrected by caesarean section. A live foal was delivered and the mare survived the surgical interference.


Response of cattle to inoculation with atypical mycobacteria isolated from soil.

Nine strains of atypical mycobacteria and a strain of the rhodochrous taxon, originally isolated from soil samples collected on the subcoastal plains of the Northern Territory, were inoculated into cattle. Each strain was injected subcutaneously into one animal and into a mesenteric lymph node of another. At 4 and 10 weeks after inoculation, the cattle were tuberculin tested with bovine PPD tuberculin, avian PPD tuberculin and the appropriate homologous PPD tuberculin. Six strains induced a significant level of sensitivity to bovine PPD at the 4-week test, but only one animal gave a similar response at the 10-week test. In general, the level of sensitivity to all tuberculins declined between the 4-week and 10-week tests. At both tests the response to avian PPD was equal to, or exceeded, that to bovine PPD. The inoculation of each of the 10 strains resulted in the production of tuberculous granulomas at the subcutaneous sites and similar lesions were produced at the mesenteric lymph node site in response to 2 strains. Mycobacteria were re-isolated from 11 cattle and represented 7 strains. The significance of the soil as a reservoir of atypical mycobacteria and other organisms capable of inducing sensitivity to bovine PPD is discussed.


The Ocean-Hill Brownsville and Cambodian-Kent State crises: a biobehavioral approach to human sociobiology.

The author traces the origin of his thinking on a biobehavioral systems approach to human sociobiology as it evolved from his observations and analyses of on-the-ground human behavior in local, Ocean-Hill Brownsville, and nationwide, Cambodia-Kent State, political crises, as well as from his naturalistic observations of face-to-face behavior in group psychotherapy. He argues that the resulting sociobiological paradigm--arrived at independently by several other workers--is a fruitful alternative to sociobiological models derived from population biology and genetics.


Responses of vessel walls to chronically applied electrical stimuli.

8 mm long sections of common carotid arteries of conscious, freely moving rabbits were electrically stimulated with DC-impulses by chronically implanted gold-electrodes daily for 1/2 h or two times 1/2 h per day for 4--6 weeks. The electrodes were arranged in such a way that the wall was stimulated transmurally, In the initial phase the tension of the vessel wall increased. After 1/2 h of stimulation the artery wall relaxed at the stimulated section. Repetition of stimuli led to smooth muscle cell proliferation within the stimulated region. The smooth muscle cells formed a cushion protruding into the lumen of the artery mainly at the anodic site of the stimulation-arrangement. The proliferating cells were smaller than those of the underlying tunica media. They were identified as smooth muscle cells by electron-microscopy, contained relatively more lysosomes and endoplasmic reticulum than the original media-cells and produced collagen-fibrils and elastic fibrils as an extracellular matrix material. Below very thick cushions of smooth muscle cells necrosis developed. Animals which were additionally fed with 2% cholesterol in normal food developed typical atheromatous plaques at the site of the anodic stimuli.


Experimental coronary artery occlusion. I. Measurement of infarct size.

We studied the size of infarcts in 25 dogs 48 hrs after proximal occlusion of the left anterior descending coronary artery. In one group of animals infarct size was measured by histologic criteria, in another group the infarct was measured macrohistochemically using p-NBT and malate to incubate unfixed slices of myocardium. In both groups infarct size was expressed as percentage of the area of perfusion of the occluded artery. Infarct size was 72% of the area-at-risk in the group studied by histology and 74.5% in the macrohistochemical group. The satisfactory agreement of both methods favors the p-NBT technique because of its ease and speed. It is suggested that the expression of infarct size as percentage of the perfusion area is a good definition and should be used in experiments designed to manipulate infarct size. In this way differences in the size of occluded arteries and their respective perfusion areas have no or only a negligible influence on infarct size.


The syndrome of absent pulmonary valve and ventricular septal defect--anatomical features and embryological implications.

Four cases of absent pulmonary valve in combination with ventricular septal defect are reported. In this syndrome hypo- and dysplasia of the pulmonary valve is constantly associated with a big ventricular septal defect, formation of a huge pulmonary artery aneurysm and absence of the ductus arteriosus. Presence or absence of a right ventricular outflow tract obstruction is the criteria for classification into two forms. Absence of the pulmonary valve, right ventricular outflow tract obstruction and a malalignment-type ventricular septal defect produced by a conotruncal malseptation process represent the primary complex of malformations. Consecutive intrauterine cardiac failure is most probably prevented by prenatal closure of the ductus arteriosus. Pulmonary artery aneurysm and also dilatation of the right ventricular outflow tract as well as a whole lot of other coexisting deformities can be explained by a cascade of hemodynamical sequelae started by this ductus closure in utero. An embryological scheme explaining the genesis of this syndrome is derived from a morphological analysis of the constituting incoherent-appearing anatomical features.


Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.


Properties of the iron--sulphur proteins of the benzene dioxygenase system from Pseudomonas putida.

A purification procedure was developed to stabilize the iron-sulphur proteins of the benzene dioxygenase system from Pseudomonas putida. The intermediate electron-carrying protein has a mol. wt. of 12300 and possesses one (2Fe--2S) cluster, whereas the terminal dioxygenase has a mol.wt. of 215300 and possesses two (2Fe--2S) clusters. The order and stoicheiometry of electron transfer and of the whole system are described.


"Affinity" chromatography of steroid-transforming enzymes with a non-steroidal ligand.

The chromatographic behaviour of an avian oestradiol-17 beta dehydrogenase, the 3(17) beta-hydroxy steroid dehydrogenase from Pseudomonas testosteroni and cortisone reductase from Streptomyces dehydrogenans was studied on columns of p-(phenoxypropoxy)aniline attached to CNBr-activated Sepharose. The ligand was effective in adsorbing the oestradiol dehydrogenase from a partially purified extract of chicken liver, and the cortisone reductase was perferentially retained when mixtures of the three dehydrogenases were applied to columns in 10mM-buffer. Under these conditions the 3(17)beta-hydroxy steroid dehydrogenase was eluted in the front, but was adsorbed in the presence of 3 M-KCl. beta-N-Acetylglucosaminidase present in the liver preparation was not retained by the ligand, whereas lactate dehydrogenase from rabbit muscle was adsorbed in a manner similar to the retention pattern found on affinity chromatography with 2',5'-ADP--Sepharose. The mean overall purification of the oestradiol dehydrogenase was 13-fold, with a mean recovery of 53%. p-(Phenoxypropoxy)aniline offers promise for the purification of steroid-transforming enzymes where elution with substrate or cofactor is not wanted. It is also suggested that the ligand may be of service in the purification of receptors of hormonal steroids.


The binding and catalytic activities of forms of ligandin after modification of its thiol groups.

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.


Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.


Triethyltin binding to cat haemoglobin. Evidence for two chemically distinct sites and a role for both histidine and cysteine residues.

Triethyltin binding to cat haemoglobin was measured after pretreatment of the protein with diethyl pyrocarbonate at pH 6.0,iodoacetamide or phenylmercuric acetate or by photo-oxidation in the presence of Methylene Blue. The pentaco-ordinate nature of the binding of triethyltin to cat haemoglobin is confirmed by the inability of intramolecularly pentaco-ordinate tin compounds to compete. Consideration of the symmetry of the haemoglobin molecule in the light of the above results suggests that a unique arrangement of histidine and cysteine residues is required for the binding of triethyltin. The effects of treatment with diethyl pyrocarbonate of other preparations which bind triethyltin (rat liver supernatant, a fraction from rat liver mitochondria and rat brain myelin) were determined and shown to be complex.


Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.


A four-straight-line model for the proteinase-binding characteristics of human blood serum.

Kinetic evaluation of the capacity of human blood serum to form complexes with bovine trypsin generated partition profiles that may be approximated by a series of four intersecting straight lines. Such profiles are suggested to reflect the binding of trypsin to alpha 2-macroglobulin in a kinetically preferred mode (alpha-sites), followed by a subsidiary mode (beta-sites) and finally to alpha 1-antitrypsin. The form of the profile, in addition to revealing a hitherto unreported proteinase-binding capability of alpha 2-macroglobulin (beta-sites), also indicates that saturation of alpha-sites corresponds to a molar binding ratio of alpha 2-macroglobulin/trypsin of 1:2. Finally the profile provides, for certain pathological states, a clinically valuable characteristic.


Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds.

The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.


Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues.

1. An acidic protein with properties similar to those of troponin C from rabbit skeletal muscle has been shown to be present in bovine and rabbit smooth muscles, chicken gizzard and rabbit liver, kidney and lung. 2. A simple new method involving the use of organic solvents is described for the purification of the troponin C-like proteins from various tissues. 3. The troponin C-like proteins can be distinguished from rabbit skeletal-muscle toponin C by their electrophoretic behaviour on polyacrylamide gels at pH 8.3 in the presence and absence of Ca2+. The troponin C-like proteins have been shown to form complexes with rabbit skeletal-muscle troponin I that migrate on electrophoresis in polyacrylamide gels. 4. Behaviour on electrophoresis, amino acid analysis and the patterns of CNBr digests on polyacrylamide gels indicate that the troponin C-like proteins from bovine uterus and aorta, rabbit uterus, and liver and chicken gizzard are very similar to, if not identical with, bovine brain modulator protein. 5. With bovine cardiac muscle the organic-solvent method yields a preparation consisting of roughly similar amounts of troponin C and troponin C-like protein. 6. By the isotope-dilution technique, troponin C-like protein has been shown to represent 0.42% of the total protein in rabbit uterus. 7. In homogenates of smooth muscle, rabbit lung, kidney and brain, the troponin C-like proteins form a complex with other protein (or proteins) that requires Ca2+ for its formation and that is not dissociated in 9M-urea.


A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods.

A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.


Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate.

1. It was proposed [Johnson (1974) J. Neurochem.23, 785-789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with (3)H and (32)P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37 degrees C occurred with a half-life of about 2-4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred (3)H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.


Additional evidence for a proform to tropoelastin from chick aorta.

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10--20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.


Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.


The reaction of rabbit muscle creatine kinase with some derivatives of iodoacetamide.

The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.


Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.


Patterns of apparent co-operativity in a simple random non-equilibrium enzyme--substrate--modifier mechanism. Comparison with equilibrium allosteric models.

It has often been claimed that random non-equilibrium mechanisms can result in apparent homotropic and heterotropic effects in steady-state kinetics of the kind more usually attributed to intersubunit allosteric interactions. However, it has never been shown whether any simple random mechanism could in fact give patterns of apparent interaction similar to those predicted by the well-known allosteric models. The patterns of apparent substrate co-operativity and affinity given by the steady-state of a standard simple random substrate-modifier mechanism in which catalytic velocity is proportional to substrate binding have been analysed mathematically and numerically. All patterns possible with this model are described. Some of them rather resemble those possible with standard allosteric models, in that there is a high-affinity and a low-affinity form at zero and infinite modifier concentrations (or vice versa) which show Michaelian behaviour, apparent co-operativity passing through a maximum or minimum at intermediate affinities. Unlike the allosteric models the family of curves is in principle not symmetrical. The random model can also give behaviour not possible with the standard allosteric models, such as higher substrate affinity at intermediate modifier concentrations than at either zero or infinite modifier, with concomitant negative apparent substrate co-operativity, or a single change of sign of apparent substrate co-operativity. The analysis uses recently discovered simplified forms of steady-state equations for random models.


Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.


Purification of the chloroplast-membrane dicyclohexylcarbodi-imide-binding proteolipid by ion-exchange chromatography.

An efficient, mild and rapid procedure is reported for the separation of the dicyclohexyl-carbodi-imide-binding protein of chloroplast membranes from endogenous lipid components. By the use of ion-exchange chromatography the chloroplast proteolipid can be successfully separated from the major part of chlorophyll and other membrane lipids while being retained in a butan-1-ol milieu.


Removal of cadmium(II) from crystallized ferritin.

The cadmium content of crystallized horse spleen ferritin, usually about 2% by weight without special treatment, can be substantially decreased by prolonged dialysis against certain chelating agents, chaotropic ions, or weakly reducing anions. For example, neutral bisulphite buffer (2M) removed 95% of the bound cadmium of crystallization without affecting the iron content, and may thus be valuable for preparing "metal-free" holoferritin for physical-chemical studies.


Ligand-induced conformational transitions and secondary structure composition of chicken liver pyruvate carboxylase.

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.


Purification and some properties of a protein factor binding and deacylating initiator transfer ribonucleic acid.

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNA(fMet) and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNA(Met) or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8-10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNA(fMet) is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNA(fMet) and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1-2mm). The ternary complex of the factor with methionyl-tRNA(fMet) and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNA(fMet) not required for translation.


Enzyme-induced inactivation of transminases by acetylenic and vinyl analogues of 4-aminobutyrate.

The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino-hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration.


The direct radioimmunoassay of oestrogen glucuronides in human female urine.

Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.


The intrinsic fluorescence of isolated central-nervous-system myelin-sheath preparations.

The intrinsic fluorescence characteristics of tyrosine and tryptophan residues in the proteins of isolated central-nervous-system myelin were investigated to gain information concerning the location of these residues within the intact membrane system. Tryptophan fluorescence from isolated myelin has an emission maximum at 325 nm that appears to arise from at least two different populations of tryptophan residues. Further evidence for heterogeneity of tryptophan location in the membrane is obtained from quenching studies with chloroform and acrylamide. It is speculated that one tryptophan population is hydrophobically situated and may be derived from the proteolipid protein of myelin, whereas the other tryptophan population is located at the membrane surface and may arise from the extrinsic basic protein. A significant tyrosine fluorescence is detected from isolated myelin, indicating that some of these residues are not quenched by structural interactions within the lipid--protein membrane system. Studies with freeze-dried resuspended myelin suggest that the structural arrangement of protein components in the dried rehydrated membrane system differs significantly from that of the freshly isolated myelin membrane.


Carbohydrate composition of human placental N-acetylhexosaminidase A and B.

The carbohydrate composition of N-acetyl-beta-D-hexosaminidases (EC 3.2.1.52) A, B and heat-converted B was determined by g.l.c. Similar quantities of mannose, N-acetyl-glucosamine and galactose are present in the A and B isoenzymes, whereas N-acetyl-neuraminic acid is found in significant amount in only the A isoenzyme. The heat-converted hexosaminidase B also contains only trace amounts of N-acetylneuraminic acid, but is about 1.5-fold richer in mannose and N-acetylglucosamine and nearly 2-fold richer in galactose than native hexosaminidase B. Since native and converted hexosaminidase B are thought to be composed of four identical protein chains, our results suggest that there may be variable glycosylation of these chains.


Subunit composition of human liver alpha-L-fucosidase.

The subunit composition of human liver alpha-L-fucosidase was studied by using three different procedures for treating and electrophoresing the purified enzyme. Although the presence on only one subunit was found by conventional procedures using sodium dodecyl sulphate and urea, two non-identical subunits with mol. wts. of 59 000 and 54 000 were found when the alpha-L-fucosidase was reduced and S-carboxymethylated with iodoacetate followed by electrophoresis on polyacrylamide gels containing 0.1% sodium dodecyl sulphate and 8.0 M-urea.


Lipid and steroid hydroperoxides as substrates for the non-selenium-dependent glutathione peroxidase.

The reduction of linoleic acid hydroperoxide catalysed by rat liver cytosol was previously shown to be catalysed by a selenium-dependent glutathione peroxidase. In contrast, the enzyme responsible in guinea-pig liver cytosol is not selenium-dependent and appears to be a glutathione transferase.


Mapping the membrane proteins of Newcastle-disease virus with a photoreactive glycolipid probe.

The envelope proteins of Newcastle-disease virus were preferentially labelled when a suspension of virus particles that contained the photoreactive probe 12-(4-azido-2-nitro-phenoxy)stearoyl[1-14C]glucosamine was irradiated. One of the proteins labelled was not readily accessible to surface labelling with 125I.


Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy.

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.


Suppression of the formation of polyamines and macromolecules by DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) in phytohaemagglutinin-activated human lymphocytes.

1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.


Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.


Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of N tau-methylhistidine.

The role of glucocorticoids in regulating the rate of muscle protein breakdown was evaluated by measuring excretion of N(tau)-methylhistidine during administration of various doses of corticosterone to adrenalectomized rats. Groups of rats received daily subcutaneous injections of 0, 0.2, 0.5, 1.0, 5.0 or 10.0mg of corticosterone/day per 100g body wt. for 7 days, followed by 3 days without hormone treatment, after which they were killed. A group with intact adrenal glands served as an additional control. All animals were pair-fed with the untreated adrenalectomized group. No significant differences were noted in growth rate or N(tau)-methylhistidine excretion between the intact or adrenalectomized control groups, or those given 0.2, 0.5 and 1.0mg of corticosterone, whereas growth ceased and N(tau)-methylhistidine excretion rose markedly in the groups receiving 5 and 10mg of corticosterone. After these two high doses of corticosterone, but not after lower doses, there was a loss of weight of the gastrocnemius muscle per 100g of final body wt., but not of the soleus and extensor digitorum longus muscles. The two highest doses of corticosterone also resulted in an increase in liver weight per 100g of final body wt. Lower doses of corticosterone did not cause these changes. Plasma corticosterone concentrations, measured on the final day of injection and again at the time of killing, were decreased to near zero by adrenalectomy and were little raised by doses of 0.2 and 0.5mg daily, but were increased to within the normal range by the 1mg dose. At 5 and 10mg doses, plasma corticosterone concentrations were sustained at 2-3 times those of intact rats, and thus in the range reported for rats exposed to severe stress. Rats given 5 and 10mg doses of corticosterone had glycosuria, and showed considerably elevated concentrations of insulin in the plasma. It is concluded that plasma concentrations of glucocorticoids within the normal range do not regulate the rate of muscle protein breakdown, whereas excessive plasma concentrations of corticosteroids, equivalent to those observed in severe stress, can accelerate muscle protein breakdown.


Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.


Computed resolution and relative specific radioactivities of radiolabelled proteins synthesized by isolated gastric mucosal cells.

1. By radiolabelling, polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography, more than 50 soluble proteins in the molecular-weight range 15000-100000 were shown to continue to be synthesized after cells had been isolated from rat gastric mucosa. 2. Densitometric measurements of stained gels and fluorographic films were processed by computer to resolve individual overlapping Gaussian peaks corresponding to the protein bands. 3. Comparison of resolved peak areas of radioactivity and staining showed certain bands to have characteristically high relative specific radioactivities. 4. The computer programs (in FORTRAN) permit the analysis of a single densitometric trace or the simultaneous comparison of a corresponding pair of densitometric records of stained gels, or of fluorographic films, or a combination. Central processing unit time is used economically. 5. The programs identify the Gaussian components that contribute to the records and estimate their means, standard deviations and enclosed areas. These estimates are improved by a piecewise iterative method that minimizes the errors between the calculated and the experimental data. 6. Relative specific radioactivities are calculated as the normalized ratio of the area of a fluorographic film peak and the area of the corresponding stained gel peak. The computer programs have been deposited as Supplementary Publication SUP 50094 (55 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1978) 169, 5.


Methane synthesis without the addition of adenosine triphosphate by cell membranes isolated from Methanobacterium ruminantium.

The membrane fraction isolated from broken cells of Methanobacterium ruminantium actively synthesized methane from CO2 and H2 without the addition of ATP or other cofactors. This activity was lost unless strictly anaerobic conditions were maintained throughout the isolation and incubation procedures. 3H2, but not 3H2O, was readily incorporated into methane. This indicates that hydrogen atoms are used in the formation of methane without the prior equilibration of protons with the water phase. Methylenetetrahydrofolate was shown to be converted into methane, but less efficiently than CO2. The evidence indicates that tetrahydrofolate derivatives may not be of primary importance in the formation of methane from CO2 and H2. No requirement for ATP in methanogenesis could be demonstrated. However, chemical reagents that can increase proton conductance in membranes and therby abolish the membrane electrical potential were also effective inhibitors of methanogenesis. It was postulated that, although the reduction of CO2 to methane by bacterial membranes may require energy derived from a transmembrane potential, this does not appear to be coupled to the intermediary synthesis of ATP.


Nucleosomes from normal and regenerating rat liver.

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.


Membranes and bile formation. Composition of several mammalian biles and their membrane-damaging properties.

The total content and profile of bile salts and phospholipids are reported for several mammalian biles. Rabbit and guinea-pig biles are characterized by high proportions of conjugated dihydroxy bile salts with respect to trihydroxy bile salts, but contain relatively little phospholipid. Both rabbit and guinea-pig biles exhibit little evidence of hepatic cell damage, even though they are able to cause membrane damage (as evidenced by lysis of human erythrocytes) at low (2--3 mM) concentrations of bile salts; this lytic behaviour is also a property of their predominant bile salts. Addition of phosphatidylcholine to the bile or bile salt is able to decrease the lytic behaviour. Perhaps the most significant observation is that these biles, and their predominant bile salts, are dramatically less lytic towards sheep erythrocytes, indicating that some factor(s) in membrane composition and structure may partly explain the resistance of membranes of the biliary tract to the presence of high concentrations of potentially membrane-damaging bile salts.


Changes in the contents of adenine nucleotides and intermediates of glycolysis and the citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle.

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.


Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes.

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.


Fungal metabolism of biphenyl.

Cunninghamella elegans grown on Sabouraud dextrose broth transformed biphenyl to produce 2-, 3- and 4-hydroxybiphenyl, as well as 4,4'-dihydroxybiphenyl as free phenols. A compound tentatively identified as 2,4'-dihydroxybiphenyl was also produced. When 4-hydroxybiphenyl or 2-hydroxybiphenyl replaced biphenyl as the substrate, C. elegans produced 4,4'-dihydroxybiphenyl and 2,5-dihydroxybiphenyl respectively. The compound identified as 2,4'-dihydroxybiphenyl was produced from both substrates. A survey of 11 species of fungi known to degrade hydrocarbons revealed two species that were comparable to C. elegans in their ability to convert biphenyl into free phenols. In addition to free phenolic metabolites, deconjugation experiments indicated that 44% of the known metabolites present in the culture filtrate were present in the form of conjugates. These results suggest that the transformation of biphenyl by C. elegans is similar to that found in mammalian systems.


Participation of quinone and cytochrome b in tetrathionate reductase respiratory chain of Citrobacter freundii.

Formate dehydrogenase, NADH dehydrogenase, a quinone and a b-type cytochrome characterized by maxima at 429 and 560 nm are shown to participate in the tetrathionate redox chain of Citrobacter.


Ca2+-stimulated ribonuclease. A new marker enzyme of differentiated rat mammary tissues.

A unique ribonuclease, active only in the presence of Ca2+, was present in lactating mammary gland and milk of the rat. This enzyme was absent from virgin-rat mammary gland and non-mammary tissues of lactating rats. The presence of moderate activity in differentiated mammary tumours, together with an increase in activity in normal tissue parelleling development of mammary function, identify this enzyme as a marker of mammary differentiation in the rat.


Adenosine, thyroid status and regulation of lipolysis.

Adipocytes from hypothyroid rats do not respond to adrenaline with increased glycerol release. Adenosine deaminase largely restores lipolytic sensitivity. This effect is reversed by 2-deoxycoformycin, an inhibitor of the enzyme, and by N6-(phenylisopropyl)adenosine, which is not deaminated. Lipolytic response of normal cells to adrenaline is only 50% inhibited by phenylisopropyladenosine, whereas in cells from hypothyroid rats blockage is total. Inhibition of 50% was seen at 100 and 1 nM concentrations respectively. Insensitivity to adrenaline of hypothyroid-rat adipocytes can, at least partly, be explained by increased sensitivity to adenosine.


L-proline transport by newborn rat kidney brush-border membrane vesicles.

The transport of L-proline was studied in brush-border membrane vesicles isolated from the kidneys of newborn rats. In contrast with the rapid initial uptake with an 'overshoot' observed in adult vesicles, uptake by the newborn vesicle was slow, showed no 'overshoot', and proline continued to accumulate at a time when the adult vesicle had already equilibrated. L-Proline transport in the newborn rat occurs by Na+-dependent and independent mechanisms. There appeared to be essentially no uptake by anti-luminal vesicles isolated from newborn rat kidney. These observations may help to explain the prolinuria that occurs in the newborn animal.


Inhibition of phosphatidylinositol synthesis and the inactivation of calcium entry after prolonged exposure of the blowfly salivary gland to 5-hydroxytryptamine.

The incorporation of [32P]Pi into all salivary-gland phospholipids except phosphatidic acid was inhibited by 5-hydroxytryptamine. The accumulation of [32P]Pi into phosphatidic acid was actually enhanced by 5-hydroxytryptamine. There was an inhibition of labelled inositol incorporation into phosphatidylinositol by 5-hydroxytryptamine, which seems to be mediated by calcium because it was mimicked by the ionophore A23187, but was prevented if glands were stimulated with 5-hydroxytryptamine in the absence of external calcium. Inhibition of synthesis together with stimulation of breakdown will decrease the concentration of phosphatidylinositol, which could account for the inactivation of calcium transport observed at high 5-hydroxytryptamine concentrations. When salivary glands were stimulated with 1 micrometer-5-hydroxytryptamine, there was a rapid increase in the transfer of 45Ca2+ from the medium into the saliva, but with time this transport declined to a low value. If the glands were washed free of 5-hydroxytryptamine and incubated in the presence of 2mM-inositol for 1 h, the increase in calcium transport caused by 5-hydroxytryptamine was restored. There was little recovery in the absence of inositol. If glands were stimulated with 5-hydroxytryptamine in the absence of external calcium, a condition which prevents the inhibition of phosphatidylinositol synthesis, calcium transport in response to 5-hydroxytryptamine was greater than in glands preincubated with 5-hydroxytryptamine in the presence of calcium. The inactivation of calcium transport may result from a decrease in phosphatidylinositol concentration. These results support the hypothesis that the hydrolysis of phosphatidylinositol plays some role in either the opening or closing of calcium 'gates'.


Nuclear and cytosolic distribution of conjugated cholic acid and radiolabelled glycocholic acid in rat liver.

1. Normally fed and cholestyramine-treated rats were injected through the superior mesenteric vein with different amounts of radiolabelled glycoholic acid and the appearance of radioactivity in bile was measured. 2. In normally fed rats radioactivity appeared in bile within 30 s of injection and reached a maximum after 2 1/2 min; in the cholestyramine-treated animals the appearance of radioactivity was slower and less of the injected material was excreted into bile. 3. At 10 min after injection, livers were removed and the amounts of radioactive glycoholic acid and endogenous cholic acid conjugates in nuclei and cytosol were determined; most of the bile acid was found in the cytosol, only small amounts being found in nuclei. 4. Nuclear preparations from both normally fed and cholestyramine-fed rats were extracted with KCl (0.4 M) in an attempt to identify a putative bile acid receptor, but no such receptor was found. 5. Regulation of bile acid synthesis does not involve nuclear binding of bile acids.


Isolation of lysosomes from brain tissue. A separation method by means of alteration of mitochondrial and synaptosomal sedimentation properties.

1. A crude mitochondrial-lysosomal preparation from brain tissue was layered on a sucrose gradient containing 20mm-succinate, 10mm-Tris and 1mm-disodium EDTA at pH7.4. The lysosomes were separated from the mitochondria and synaptosomes by means of a twosteps centrifugation procedure. In a first low-speed step (40min at 5300g at 15 degrees C) the sedimentation rate of mitochondria and mitochondria-containing synaptosomes was enlarged due to passage of these subcellular structures through the sucrose gradient with the above-mentioned chemicals (called ;chemical field'). That part of the gradient which contained the mitochondria and synaptosomes was removed and substituted by a gradient suitable for isopycnic isolation of lysosomes in a second centrifugation step. The achieved purification for bovine brain lysosomes was 5-8-fold, for rat brain lysosomes 7-10-fold, over the homogenate. 2. The enlargement of the sedimentation rate of mitochondria and synaptosomes was brought about by the presence of succinate, but also by one of the following salts in the chemical field: malonate, fumarate, pyruvate, phosphate and chloride. 3. Comparison of the chemical-field method with other methods for the isolation of lysosomes showed that (a) with the chemical-field method a 2-3-times higher purification of the rat and bovine brain lysosomal fraction can be achieved than with the procedure described by Koenig, Gaines, McDonald, Gray & Scott [(1964) J. Neurochem.11, 729-743], and that (b) similar purification results for rat liver lysosomes were obtained when the chemical-field method and the procedure described by van Dijk, Roholl, Reijngoud & Tager [(1976) FEBS Lett.62, 177-181] were compared.


The distribution of enzyme and isoenzyme activities between parenchymal and haematopoietic cells in the liver of the foetal guinea pig.

The hepatocyte and haematopoietic cell contents of the liver of the foetal guinea pig were measured over the latter half of gestation. Hepatocytes represented about 30% of liver volume at mid-gestation and this increased to 70-80% by term; cell volume remained fairly constant until 5-7 days before term, then more than doubled. Haematopoietic cells represented about 5% of liver volume at mid-gestation and this progressively fell to <1% by term. At 75% of gestation hepatocytes and haematopoietic cells were prepared from perfused foetal livers by collagenase digestion. Enzyme activity of the hepatocyte was, without exception, similar to that of the whole liver. In general, enzyme activity in the haematopoietic cells was similar to that in erythrocytes, with relatively low values for aldolase, glycerol 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, lactate dehydrogenase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, isocitrate dehydrogenase, ;malic' enzyme, glutamate dehydrogenase and aspartate aminotransferase. The haematopoietic cell contribution to total enzyme activity in the foetal liver was usually much less than 10% and could thus not account for the major changes in hepatic enzyme activity over the latter half of gestation. Hepatocytes contained hexokinase isoenzymes I and III, aldolase isoenzymes A and B and pyruvate kinase isoenzymes 1, 2 and 4. The haematopoietic cells contained hexokinase isoenzyme I and two additional bands of activity with slightly greater mobility, aldolase isoenzyme A and pyruvate kinase isoenzymes 2 and 4.


The effects of barbiturates on the metabolism of phosphatidic acid and phosphatidylinositol in rat brain synaptosomes.

Barbiturates and diphenylhydantoin inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol and phosphatidic acid, but have a relatively slight effect on the incorporation of 32P into these lipids in the absence of carbamoylcholine and no effect on 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. Inhibition of the carbamoylcholine-stimulated increase was observed for pentobarbital, thiopental, phenobarbital, 5-(1,3-dimethylbutyl)-5-ethylbarbiturate, (+)- and (-)-5-ethyl-N-methyl-5-propylbarbituate and diphenylhydantoin. Similar concentrations of barbiturates and diphenylhydantoin were previously reported to inhibit the K+-stimulated Ca2+ influx, and therefore other agents that affect Ca2+ influx were tested to find whether they had any effect on 32P incorporation into these lipids. K+ (35 mM) increases 32P incorporation into phosphatidic acid, but to a smaller degree than 100 micrometer-carbamoylcholine, and its effect was inhibited by pentobarbital. Veratridine (75 micrometer) does not increase 32P incorporation into either phosphatidic acid or phosphatidylinositol, but did inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol. The possible relationship between the phospholipid effect and stimulated Ca2+ influx is discussed.


High-performance liquid chromatographic separation and identification of epimeric 17-ketone impurities in commercial sample of dexamethasone sodium phosphate.

A commercial sample of dexamethasone sodium phosphate solution for injection was found to contain 56% of the label concentration and to be extensively contaminated (approximately 50%) with a white insoluble solid, which was identified as a mixture of the 16 alpha- and 16 beta-methyl epimers of 9-fluoro-11 beta-hydroxy-16-methylandrosta-1,4-diene-3,17-dione. High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate these epimers and to determine their presence in commercial samples. One epimer was identified by HPLC comparison with a synthesized specimen of 9-fluoro-11 beta-hydroxy-16 alpha-methylan-drosta-1,4-diene-3,17-dione. The second peak was identified as the 16 beta-epimer by epimerization of the synthesized alpha-component with alkali to obtain a product whose chromatogram matched that of the impurity. These conclusions are supported by data obtained by IR and UV spectrophotometry, TLC, and the blue tetrazolium test.


Decreased tetracycline bioavailability caused by a bismuth subsalicylate antidiarrheal mixture.

Oral coadministration of a single 250-mg tetracycline capsule and 60 ml of a bismuth subsalicylate antidiarrheal mixture reduced tetracycline absorption by 34% without appearing to perturb its absorption or disposition rate. A pronounced increase in intersubject tetracycline absorption variability also was noted. Apparently, the reduction in tetracycline bioavailability previously reported with a kaolin-pectin suspension is not peculiar to kaolin-pectin but can be expected with almost any antidiarrheal whose mechanism of action is adsorptive in nature.


Effect of phosphorus depletion on intestinal calcium and phosphorus absorption.

Intestinal calcium (Ca) hyperabsorption is a well-documented feature of experimental phosphorus depletion (PD). To further evaluate the effect of PD on Ca absorption we studied metabolic balance and in vitro everted duodenal sac uptake of Ca and phosphorus (P) in weanling male rats. Animals were assigned to three dietary groups: normal, 0.3% P ad libitum (NP); low, 0.03% P ad libitum (LP); and normal, 0.3% P but pair-fed with assigned LP mates (NP-PF). Results indicate that although PD led to an early but unsustained increase in 45Ca uptake by the everted duodenal sac in vitro, net intestinal Ca retention is consistently decreased in rats on the LP diet compared with rats eating either the NP or NP-PF diet. The reduction in net intestinal Ca absorption is reflected by an increase in fecal Ca, both in absolute quantities and in proportion to dietary Ca intake. The initial negative P balance after the initiation of the LP diet was promptly, albeit precariously, corrected. This was associated with a sustained increase in duodenal 32P uptake in vitro and virtual cessation of growth. Because the biosynthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its accumulation in intestinal mucosa have been reported to increase with PD, our study represents an example in which the physiological interrelationship between the activity of 1,25(OH)2D3 and intestinal Ca absorption may be dissociated.


Identification of susceptibility to malignant hyperpyrexia in swine.

In vitro muscle contracture responses in swine susceptible to malignant hyperpyrexia (MH) were similar to those found in muscle from humans susceptible to this anaesthetic complication, confirming the suitability of the pig as an animal model for studying MH. The results suggest that there are different degrees of susceptibility to MH. Whichever drug was used, there was some overlap in the contracture responses between susceptible animals and controls, suggesting that the most accurate way of identifying susceptibility to MH is to use a variety of chemical agents, the best of which seem to be halothane, caffeine, suxamethonium and potassium chloride. Thymol, which is used as a preservative in commercial preparations of halothane, potentiates halothane contractures, but it is not known if this is significant clinically.


Threshold concentration of nitrous oxide affecting psychomotor performance.

Using audiovisual reaction times, no effects were found in 12 subjects exposed to 1, 2, 4, or 8% nitrous oxide. In subsequent studies on 30 subjects, a positive effect on performance was found at a concentration of between 8 and 12% nitrous oxide. In addition, there was no difference in mean reaction time in 12 subjects exposed to air or 8% nitrous oxide. It is concluded that the threshold concentration of nitrous oxide for an effect on psychomotor performance as assessed by choice reaction times probably lies between 8 and 12%.


In vitro interaction of propanidid and suxamethonium with pooled human plasma cholinesterase. A kinetic study.

Human plasma cholinesterase (E.C. 3.1.1.8) was shown to be inhibited by physiological concentrations of propanidid and suxamethonium using a colourimetric assay at 25 degrees C and pH 7.2 unit with butyrylthiocholine as substrate. Propanidid inhibited the enzyme in a non-competitive manner (I50 = 2.0 mmol litre-1; apparent Km = 6.6 X 10(-4) mol litre-1) as did suxamethonium (I50 = 4.4 mmol litre-1; apparent Km = 1.6 X 10(-4) mol litre-1). Combined inhibition produced Km 3.0 X 10(-3) mol litre-1. The binding of these drugs to specific anionic sites in the vicinity of the active centre is thought to result in stereochemical changes in the enzyme. This mechanism and its relevance to the augmentation of the neuromuscular blockade produced by suxamethonium in the presence of propanidid is discussed.


Failure of clinical concentrations of halothane to block adenosine diphosphate-induced platelet aggregation in vitro.

A dose-related inhibition by halothane of adenosine diphosphate-induced platelet aggregation was measured for human platelets in plasma anticoagulated with sodium citrate 6.8 mmol litre-1 or 13.6 mmol litre-1, or heparin 2 u. ml-1 and for platelets separated from plasma by albumin density gradient centrifugation and requiring no anticoagulant. By maintaining exposure and assay temperature at 37 degrees C, maintaining physiological pH, avoiding age-related deterioration and using an atraumatic equilibration method, no inhibition was found at clinical concentrations of halothane and 50% inhibition required greater than halothane 10%. Sodium citrate, a calcium chelating anticoagulant, was found to enhance halothane inhibition.


Canine coronary blood flow responses to hypoxaemia: the influence of halothane.

Electromagnetic flow meters were used to measure blood flow in the coronary and pulmonary arteries of six anaesthetized dogs following thoracotomy. Halothane 1% (v/v) caused a reduction in arterial pressure, coronary artery flow, cardiac output and myocardial and total body oxygen consumption and an increase in coronary vascular resistance. Hypoxaemia caused large increases in coronary artery flow during both cardiac systole and diastole, but not until PaO2 was less than 5.3 kPa. This response was not influenced by the presence of halothane. Although myocardial oxygen availability and consumption were maintained during hypoxaemia, total body oxygen availability and consumption were markedly reduced.


Pulmonary mechanics during general anaesthesia. The influence of mechanical irritation on the airway.

The changes in pulmonary resistance (RL) and compliance (CL), following airway irritation and surgical stimulation, were investigated in 19 anaesthetized, paralysed patients. Thirteen were normal, while six had objective evidence of chronic obstructive pulmonary disease (COPD). Broncho-carinal irritation with a suction catheter produced a 27% increase in RL (0.58+/-0.32kPa litre-1 s to 0.74+/-0.40, P less than 0.01) and a 10% decrease in CL (0.87+/-0.19 litre kPa-1 to 0.81+/-0.22, P less than 0.01). These changes were associated with a significant increase in systolic arterial pressure and heart rate during clinical stages of anaesthesia (end-tidal enflurane, 1.3+/-0.4%, PaCO2 5.20+/-0.53 kPa). These changes did not correlate with depth of anaesthesia, but all returned to the pre-irritation value within 5 min. There was no difference in subjects considered normal and those with COPD. Surgical stimulation did not produce significant changes in pulmonary mechanics; however, repeated broncho-carinal irritation during surgery was associated with a transient increase in RL (P less then 0.01).


A new method for measuring PCO2 during anaesthesia.

We have evaluated a new method for measuring the oxygenated mixed venous PCO2 (PVCO2) in patients undergoing general anaesthesia. The lungs were inflated with a gas mixture containing 0% carbon dioxide and then 12% carbon dioxide and the expired gas was analysed both before and after a brief period of breath-holding. PVCO2 was estimated from the differences in carbon dioxide concentration before and after the apnoeic period. Simultaneous measurments of PaCO2 were made in blood obtained from radial artery puncture. The range of PaCO2 studied was 3.2--6.13 kPa. The relationship between mixed venous and arterial PCO2 was found to be PaCO2 = 0.87 PVCO2--0.44 (r = 0.91). We conclude that this method for measuring PVCO2 can be used during anaesthesia allowing PaCO2 to be estimated with considerable accuracy.


Atropine reversal of hypercarbia during enflurane anaesthesia.

Atropine was given i.v. to 35 spontaneously breathing healthy adults anaesthetized with enflurane to determine if it would reduce PaCO2 by improving gas exchange. Five minutes after administration of atropine, PaCO2 decreased from mean 7.18 to 6.65 kPa (-7%), while mean VE increased from 4.8 to 6.1 litre min-1 (+27%). These changes were maintained 15 min later in the 20 patients studied at that time. Older and more obese patients showed a more significant change. Respiratory frequency and PaO2 did not alter.


Cyclopropane and Caesarean section.

In a prospective study 589 patients were anaesthetized by the inhalation of cyclopropane 40% for 2--3 min. Relaxation was provided with suxamethonium and anaesthesia was maintained with cyclopropane 7.5%. The series was divided into four groups: patients in optimal condition (96 elective (group 1), 164 emergency (group 3)) and those not in optimal condition (75 elective (group 2), 254 emergency (group 4)). All infants from groups 1 and 3 had high Apgar scores, and all survived. Infants in groups 2 and 4 had lower Apgar scores, eight and 96 respectively were depressed, and 10 and 20 died because of obstetric and neonatal factors unrelated to the anaesthesia. Respiratory distress syndrome (RDS) developed in 44 infants. Among 29 infants in whom the birth weight was less than 2.5 kg the mortality was 31%, whereas no death occurred in mature infants. RDS was significantly more frequent after elective than after emergency surgery. Neonatal depression was independent of the length of the induction-delivery interval, which averaged 13.3 min. The frequency of awareness was 1.5%.


Hypotensive anaesthesia for craniectomy in infancy.

Deliberate hypotension using halothane and controlled ventilation without positive end-expiratory pressure was employed in 18 of 30 patients undergoing craniectomy for unilateral or bilateral craniosynostosis over a 3-yr period. The technique was simple, resulted in good control of arterial pressure and diminished blood loss, and did not involve extensive monitoring or the use of potentially toxic adjuvant drugs. In the hypotensive group mean systolic arterial pressure was decreased from 92.5 to 65.0 mm Hg. Estimated blood loss was decreased from 111 to 89 ml (mean) for all ages and from 133 to 72 ml (mean) for infants between 8 and 32 weeks of age compared with the normotensive control group.


Metallized plastic sheeting in the prevention of hypothermia during neurosurgery.

Metallized plastic sheeting (m.p.s.) was found to be ineffective in the prevention of hypothermia in adult patients studied during neurosurgical operations. Twenty patients were wrapped in m.p.s. and 22 patients acted as controls. Mean oesophageal temperature in the m.p.s. group decreased from 36.2 degrees C to 35.4 degrees C after 3 h (control 36.5 degrees C to 35.4 degrees C). It was concluded that active warming systems are needed to maintain normothermia in patients undergoing neurosurgical operations.


Suxamethonium infusion rate and observed fasciculations. A dose-response study.

Suxamethonium chloride (Sch) was administered i.v. to 36 adult males at six rates: 0.25 mg s-1 to 20 mg s-1. The infusion was discontinued either when there was no muscular response to tetanic stimulation of the ulnar nerve or when Sch 120 mg was exceeded. Six additional patients received a 30-mg i.v. bolus dose. Fasciculations in six areas of the body were scored from 0 to 3 and summated as a total fasciculation score. The times to first fasciculation, twitch suppression and tetanus suppression were inversely related to the infusion rates. Fasciculations in the six areas and the total fasciculation score were related directly to the rate of infusion. Total fasciculation scores in the 30-mg bolus group and the 5-mg s-1 and 20-mg s-1 infusion groups were not significantly different.


Recordings of, and an anatomical explanation for, false positive loss of resistance during lumbar extradural analgesia.

Loss of resistance dorsal to the ligamentum flavum was recorded in 10 of 16 patients aged 43--75 yr whilst performing extradural anaesthesia at the L2--3 interspace via the midline approach. Degeneration of the interspinous ligament with cavity formation is observed frequently in the elderly and may account for false positive loss of resistance and failure to obtain analgesia following the injection of local anaesthetic.


Bedside angiography of the lung by Swan-Ganz catheter. A case report.

A Swan-Ganz catheter, employed for haemodynamic monitoring, was put to a use not described previously. Pulmonary angiography was performed through the catheter at the bedside with minimal discomfort or complication. In this case it was possible to determine that a lung abscess was responsible for a translucency in the right lower lobe. This was subsequently confirmed at postmortem examination.


Carcinoembryonic antigen in serum of unselected breast-cancer patients and of non-hospitalized controls.

A series of consecutive unselected patients with primary breast carcinoma and their age-matched controls were studied for serum CEA in relation to clinical findings. Raised CEA was found in a similar frequency in patients with primary breast cancer (pre- and postoperative) and in the control women: 16%, 11% and 11%, respectively, exceeded the selected upper limit of the reference range (13 ng/ml) with a double-antibody radioimmunoassay. In the breast-cancer patients, however, 48% of the raised CEA levels exceeded 16 ng/ml, compared with only 20% in the controls. Significant correlations (r approximately 0.3) were found between CEA levels and tumour size, TNM classification and a combined clinical and histopathological classification. A high frequency of raised CEA values in the advanced breast-cancer patients was the essential contribution to these positive correlations. A correlation coefficient of 0.6 was found between pre- and postoperative CEA values. The frequency of smoking and/or chronic disease was unexpectedly high in patients as well as in controls with high CEA.


Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes.


Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation.

The S-phase-specific cytotoxicity of hydroxyurea (HU) was tested on synchronized murine fibrosarcoma (FSa) cells lodged in the lungs of C3Hf/Bu mice. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations. After elutriation, 8000 viable FSa cells from each fraction, along with 10(6) heavily irradiated tumour cells (unseparated) were injected i.v. into whole-body-irradiated mice (20 per group). Under these conditions, 95% of the injected cells, regardless of size or position in the cell cycle, are arrested in the lungs. Twenty minutes later, hydroxyurea (HU, 1 mg/g) was administered i.p. into 10 animals of each group. Fourteen days later the animals were killed, their lungs removed and fixed, and the number of macroscopic tumour nodules counted. Killing of the initially injected cells by HU, as evidenced by a reduction in lung colonies in treated animals, correlated with the precentage of S-phase cells in each fraction. The greatest effect, an 80% reduction in colony number, was seen in Fraction 8, containing the largest percentage of S-phase cells (65%). These results demonstrate the usefulness of this procedure as a rapid method for characterizing the phase specificity of chemotherapeutic drugs in vivo.


Changes in nerve conduction velocity in the mouse after acute and chronic administration of nitroimidazoles.

The effect of the nitroimidazoles misonidazole, Ro-05-9963, RGW-608 and metronidazole on nerve conduction velocity (NCV) were measured in the anaesthetized mouse. The compounds were administered by i.p. injection either as a single dose of 1 mg/g (only 0.5 mg/g for RGW-608) or in 36 fractions of 0.15 mg/g over 18 days (only 4 fractions in 2 days for RGW-608). After single doses a reduction in nerve conduction velocity was seen with all the compounds except metronidazole, which had no significant effect. During chronic exposure, a reduction in NCV occurred towards the end of the course of injections. All compounds produced an effect, although RGW-608 was the most neurotoxic, giving the largest reduction in NCV after only 4 injections. After the end of chronic exposure to misonidazole, Ro-05-9963 and metronidazole, recovery to normal took 2-3 weeks.


Three years' experience with Ch1VPP (a combination of drugs of low toxicity) for the treatment of Hodgkin's disease.

In 3 years, 118 patients with Hodgkin's disease have completed chemotherapy with chlorambucil, vinblastine, procarbazine and prednisolone (Ch1VPP). The complete remission rates were 90% for 29 patients previously treated with radiotherapy, 67% for 73 patients previously untreated and 44% for 16 patients with prior chemotherapy. The 3-year survival rates for the first 70 patients in the series were 83% for previously irradiated patients, 84% for previously untreated patients and 67% for those with prior chemotherapy. Forty-seven previously untreated or previously irradiated patients in this group achieved complete remission. The 3-year disease-free survival rates for these patients were 71% and 67% respectively. This regimen gives complete remission and survival rates comparable with results obtained with combinations including nitrogen mustard, while producing fewer side-effects.


Bronchial cancer in Hong Kong 1976-1977.

Bronchial cancer is a disease of high and increasing annual incidence in Hong Kong, especially in women, whose age-specific death rates from this cause are amongst the highest in the world. A case-control study of the relationship of bronchial cancer with smoking was carried out during 1976--77, taking particular note of the histological type of the tumour. Two hundred and eight male and 189 female patients were interviewed, covering about half the total number of cases of bronchial cancer registered as dead from the disease in Hong Kong during the period of the survey. The association with smoking was more evident in males than in females, and in squamous and small-cell types, as a group, than in adenocarcinoma. Forty-four per cent of the women with bronchial cancer were non-smokers, their predominant tumour being adenocarcinoma, and in them on association could be detected with place of residence or occupation. There was no strong evidence of an association with the use of kerosene or gas for cooking; 23 did not use kerosene. The cause of the cancer in these non-smoking women remains unknown.