Interaction of difluoro-oxaloacetate with aspartate transaminase.

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.


The proteins in the Z line of insect flight muscle.

Z discs were isolated from Lethocerus flight muscle by removing the contractile proteins from myofibrils with a solution of high ionic strength. The protein composition of the Z discs was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the major proteins were alpha-actinin, actin and tropomyosin. Z lines were selectively removed from intact myofibrils by digestion with crude lipase and chymotrypsin, but not by purified lipase.


The isolation of minimally degraded hyaluronate from rat skin.

Hyaluronate was isolated quantitatively from fresh rat skin by homogenization in an Edebo [J. Biochem. Microbiol. Technol. Eng. 2, (1960) 453-479] press, extraction with buffered saline, selective elution from DEAE-cellulose, and gel chromatography on Sephadex G-200. Traces of a protein contaminant remained.


Molar absorption coefficients of porphyrin esters in chloroform determined by copper titration.

Chromatographically pure porphyrin esters free from metalloporphyrins were titrated with Cu2+ to determine the molar amount of porphyrin present. The end point of the titration was defined by a t.l.c. method detecting traces of metal-free porphyrin ester in admxture to its copper complex. From spectrometric measurement at the Soret maximum and the molar amounts found by the titration, epsilonM was calculated for proto-, copro-, penta-, hexa- and hepta-carboxylic and uro-porphyrin permethyl esters. The values for uroporphyrin showed perfect agreement with those of previous workers, whereas those for coproporphyrin were about 5% lower and those for protoporphyrin more than 20% lower. The implications of the findings are discussed. Determinations of epsilonM of some higher esters (ethyl to pentyl) and some partial methyl esters with one carboxyl group free are also presented.


Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.


Factors influencing the activity of ornithine aminotransferase in isolated rat liver mitochondria.

1. The characteristics of ornithine catabolism by the aminotransferase pathway in isolated mitochondria were determined. 2. Ornithine synthesis from glutamate and glutamate gamma-semialdehyde produced by the oxidation of proline was studied. No ornithine was formed in the absence of rotenone. 3. The mechanism of ornithine transport was reinvestigated, and the existence of an ornithine+/H+ exchange system postulated. 4. The kinetics of ornithine transport, ornithine catabolism in intact mitochondria and ornithine aminotransferase activity in solubilized mitochondria were compared. It is concluded that ornithine aminotransferase activity in liver mitochondria is rate-limited by the transport of ornithine across the mitochondrial membrane, and that this enzyme is involved primarily in ornithine degradation rather than ornithine synthesis.


Polyamines and their biosynthetic decarboxylases in various tissues of the young rat during recovery from undernutrition.

1. Weanling male and female rats were undernourished for 4 weeks and then rehabilitated by allowing ad libitum feeding. 2. During rehabilitation polyamine-biosynthetic enzymes were examined in the liver, spleen and quadriceps and gastrocnemius muscles. 3. During the first few hours of rehabilitiation there was a marked increase in liver weight, accompanied by a very marked increase in ornithine decarboxylase activity. Increases in the activity of this enzyme in other tissues did not occur until between 2 and 7 days of rehabilitation, at which time there were further increases in enzyme activity in the liver. 4. S-Adenosylmethionine decarboxylase activity also showed marked fluctuations in activity in all the tissues examined. 5. Hepatic putrescine and spermidine concentrations also varied during rehabilitation, but permine concentration remained relatively constant. Both spermine and spermidine were at normal concentrations in the liver from the 10th days of rehabilitation onwards. 6. In all of the tissues examined there were marked sex differences in the parameters studied, particularly in splenic and muscular ornithine decarboxylase activity. 7. In the tissues of the male rats, changes in polyamine synthesis paralled changes in nucleic acid and protein synthesis.


Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein.

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].


Age-related changes in protein turnover and ribonucleic acid of the diaphragm muscle of normal and dystrophic hamsters.

Diaphragm muscles of dystrophic hamsters were found to be larger than those of control animals at two of three ages studied. The additional growth of these afflicted muscles correlated with large increases in protein synthesis and concentrations of RNA. Protein breakdown was also increased in the dystrophic muscles, but to a smaller extent than synthesis.


The phosphorylation of an acidic protein of the large ribosomal subunit of Krebs II ascites cells.

The ribosomes of Krebs II ascites cells contain an acidic protein, apparently analogous to proteins L7/12 of Escherichia coli. When ascites cells were incubated with [32P]Pi, this protein became labelled, indicating that it is a phosphoprotein.


The turnover of hamster fibroblast lysosomal beta-D-glucuronidase.

The half-life of hamster fibroblast beta-D-glucuronidase (EC 3.2.1.31) was estimated to be 4-5 days by measuring the decay with time of the radioactivity in beta-D-glucuronidase isolated from cells grown in the presence of 14C-labelled amino acids. A new affinity-chromatographic procedure for the purification of beta-D-glucuronidase is described.


The effects of inhibition of gluconeogenesis in suckling newborn rats.

Inhibition of gluconeogenesis with 3-mercaptopicolinate in suckling newborn rats caused a fall in blood [glucose], but no change in their high plasma [free fatty acid] and blood [ketone bodies]. Active gluconeogenesis seems to be more important than sparing of glucose by high concentrations of fat-derived substrates for the maintenance of normal blood [glucose] in suckling newborn rats.


Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action.

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.


Substrate specificity of amino acid transport in sheep erythrocytes.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.


Deoxyribonucleic acid and polyamine synthesis in rat ventral prostrate. Effects of age of the intact rat and androgen stimulation of the castrated rat with testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 beta, 17 beta-diol.

The relationship between polyamine synthesis, growth and secretion in vivo was examined in ventral prostates from: (a) intact rats aged 3-60 weeks; (b) animals castrated for 7 days before injection with 5 alpha-dihydrotestosterone (17 beta-hydroxy-5-alpha-androstan-3-one), testosterone and 5 alpha-androstane-3 beta, 17 beta-diol for up to 10 days; (c) rats injected with the 3 beta, 17 beta-diol immediately after castration. Ornithine decarboxylase activity and the concentrations of putrescine, spermidine and spermine were measured. DNA-synthetic activity was monitored by measuring [125I]iododoxyuridine incorporation. An enhanced spermidine/spermine molar ratio reflected increased activity of the prostate. The ratio was higher (greater than 2) in prostates from sexually immature animals, than in the intact adult (1.5), suggesting that the ratio was indicative of the proliferative activity of the tissue. However, in the androgen-stimulated castrated rat, enhanced spermidine/spermine ratios tended to correlate with hypertrophy and secretion. In both sets of experiments there was a linear relationship between protein and spermidine content. High spermidine/spermine molar ratios were the consequence of a relatively low rate of accumulation of spermine relative to spermidine and protein. The relationship between polyamine synthesis and DNA-synthetic activity was investigated in cultured prostate. A combination of insulin (3 mug/ml) and testosterone (0.1 muM caused a stimulatory response in the incorporation of [125I]iododeoxyuridine and in cell division, despite a depleted polyamine content and low ornithine decarboxylase activity in the cultured tissue.


The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or  2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.


Modification of erythrocyte membranes by a purified phosphatidylinositol-specific phospholipase C (Staphylococcus aureus).

A phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was purified by a three-step procedure. The specific activity of the purified enzyme was approx. 6000 times that of the culture supernatant, with an overall recovery of approx. 10%. Estimation of the molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by gel filtration gave values of 33000 and 20000 respectively. A thiol group appears to be necessary for the activity of the enzyme. The purified enzyme had no detectable delta-haemolytic activity and was unable to hydrolyse S. aureus phospholipids. Phosphatidyl-inositol in erythrocyte 'ghosts' was readily hydrolysed by the purified phospholipase C. However, in contrast with our previous preliminary observations, phosphatidylinositol in intact erythrocytes was not significantly hydrolysed. These results suggest that at least 75-80% of the phosphatidylinositol is located at the inner leaflet of the membrane.


Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf.

1. When [2-3H]glycerol was supplied to developing maize-leaf laminae, label entered 3-sn-phosphatidycholine at a linear rate essentially from zero time, whereas other lipids were labelled at accelerating rates. On transfer of laminae from [3H]glycerol to unlabelled glycerol, radioactivity was rapidly lost from 3-sn-phosphatidylcholine and accumulated in other lipids, principally monogalactosyl diacyglycerol. 2. Degradation of these lipids showed that 3H was present only in the glycerol moiety of the lipids. 3. In double-labelling pulse-chase experiments with [14C]acetate, which labelled essentially only fatty acids and [3H]glycerol similar amounts of 14C and 3H radioactivity were lost from 3-sn-phosphatidylcholine and accumulated by monogalactosyl diacylglycerol. 4. The different molecular species of both lipids isolated from laminae during a double-labelled pulse-chase study were separated by argentation t.l.c., and the changes in the amount of radioactivity and the 14C/3H ratio in different species were compared. The greatest loss of radioactivity during the period in unlabelled substrates occurred from the 3-sn-phosphatidylcholine species containing oleate and from the dilinoleate species, and radioactivity accumulated by monogalactosyl diacyglycerol was mainly in the dilinolenate species. However, despite the considerable change in the radioactivity in these species during the chase, the 14C/3H ratio in each of them remained relatively unchanged. 5. It is proposed that 3-sn-phosphatidylcholine in the developing leaf may serve as a donor or linoleate-containing diacyl-glycerols which are incorporated into other lipids, principally monogalactosyl diacylglycerol.


The formation of menaquinone-4 (vitamin K) and its oxide in some marine invertebrates.

Three echinoderms, a crustacean, a mollusc and a coelenterate were injected with either [2-14C]mevalonate and unlabelled menadione (2-methyl-1,4-naphthaquinone) or 2-[14C]methyl-1,4-naphthaquinone. In each case the label was incorporated into menaquinone-4.[2-14C]Mevalonate was incorporated into menaquinone-4 in the crab (Carcinus maenas) only in the presence of menadione, the organism presumably being unable to synthesize the naphthaquinone nucleus. The starfish Asterias rubens and C. maenas incorporated the label into a compound which was identified chromatographically as 2,3-epoxymenaquinone-4. The significance of menaquinone-4 and these animals is discussed.


Intramitochondrial localization of the 4-aminobutyrate-2-oxoglutarate transaminase from ox brain.

In order to determne the intramitochondrial location of 4-aminobutyrate transaminase, mitochondria were prepared from ox brain and freed from myelin and synaptosomes by using conventional density-gradient-centrifugation techniques, and the purity was checked electron-microscopically. Inner and outer membranes and matrix were prepared from the mitochondria by large-amplitude swelling and subsequent density-grient centrifugation. The fractions were characterized by using both electron microscopy and different marker enzymes. From the specific activity of the 4-aminobutyrate transaminase in the submitochondrial fractions it was concluded that this enzyme is associated with the innter mitochondrial membrane.


Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.


Comparative study of nuclear binding sites for oestradiol in rat testicular and uterine tissue. Determination of low amounts of specific binding site by an [3H] oestradiol-exchange method.

1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.


Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide.

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as 'protein 21"). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as "protein 33"), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.


Microbiological degradation of bile acids, further degradation of a cholic acid metabolite containing the hexahydroindane nucleus by Corynebacterium equi.

1. The further degradation of a cholic acid (I) metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IIa), by Corynebacterium equi was investigated. This organism effected ring-opening and gave (4R)-4-[2alpha-(2-carboxyethyl)-3beta-(3-carboxypropionyl)-2beta-methylcyclopent-1beta-yl]valeric acid (VI). The new metabolite was isolated as its trimethyl ester and identified by partical synthesis. It was not utilized by C. equi. 2. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-decahydro-8abeta-methyl5-oxa-6-oxoazulen-1beta-yl]valeric acid (IVa), which is a hypothetical initial oxidation product in the above degradation, was not converted by C. equi into the expected metabolite (VI), but into 3 - [2beta - [(2S) - tetrahydro - 5 - oxofur - 2 - yl] - 1beta - methyl - 5 - oxocyclopent - 1alpha - yl]-propionic acid (VIII), the structure of which was established by partial synthesis. 3. Both the possible precursors of the metabolite (VI), an isomer of the epsilon-lactone (IVa), the gamma-lactone (XIa), and the open form of these lactones, the hydroxytricarboxylic acid (V), were also not utilized by C. equi. 4. Under some incubation conditions, C. equi also converted compound (IIa) and 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb) into 5-methyl-4-oxo-octane-1,8-dioic acid (III), (4R)-4-(2,3,4,6,6abeta,7,8,9,9aalpha,9bbeta-decahydro-6abeta-methyl-3-oxo-1H-cyclopenta[f]quinolin-7beta-yl)valeric acid (VII) and probably a monohydroxy derivative of compound (IIa) and compound (III), respectively. 5. The possibility that an initial step in the degradation of compound (IIa) by C. equi is oxygenation of the Baeyer-Villiger type, yielding compound (IVa), is discussed. Metabolic pathways of compound (IIa) to compounds (III), (VI), (VII) and (VIII) are also considered.


The release of alanine by rat diaphragm muscle in vitro.

Alanine release by rat diaphragm muscle in vitro is stimulated by glutamate, valine, leucine and glucose. The stimulation by glutamate and valine (but not leucine) is inhibited by 3-mercaptopicolinate. These results suggest a metabolic route involving phosphoenolpyruvate carboxykinase which directs amino acid carbon skeletons towards pyruvate synthesis for alanine formation.


Re-evaluation of amino-oxyacetate as an inhibitor.

Data are provided which indicate that pyruvate and/or acetaldehyde can reverse the inhibition of alanine aminotransferase and aspartate aminotransferase by amino-oxyacetate. It was shown that acetaldehyde could reverse the inhibition of gluconeogenesis from alanine and that pyruvate could reverse the inhibition of urea synthesis by amino-oxyacetate.


Long-chain acyl-coenzyme A synthetase activity of spinach chloroplasts is concentrated in the envelope.

Purified chloroplasts were disrupted and then fractionated by discontinuous sucrose-density-gradient centrifugation. Envelopes contained long-chain acyl-CoA synthetase at a specific activity 80 times the activity in the lamellae or the stroma. Acetyl-CoA synthetase was concentrated in the stroma, and chlorophyll was confined to the lamellae membranes. Phospholipase D was not detected in any fraction.


Lipoprotein lipase activity at onset of development of white adipose tissue in newborn rats.

The low triacylglycerol concentration in inguinal tissue of newborn rats did not change during the first 6h after birth, despite the relatively high lipoprotein lipase activity in the tissue. Subsequently triacylglycerol concentration and enzyme activity rose in parallel. The results show that lipoprotein lipase activity was present in the tissue before fat accumulation.


Changes in protein kinase activity in rat testes during development.

Protein kinase activity in rat testes remained fairly constant from day 16 1/2 of embryonic life up to 10 days after birth. At the 21st postnatal day a nadir of activity was observed, and after an increase at 35 days of age a decrease in activity at 60 days was seen. The enzyme reached maximal specific activity in the testes of 90-day-old rats.


Prenatal induction of sucrase activity in rat jejunum.

Daily application of cortisone acetate (10mg/100g body wt.) or L-tri-iodothyronine (20 microng/100g body wt.) to female rats in the last (third) week of pregnancy elicits a precocious appearance of jejunal sucrase in their foetuses.


Correlation between levels of breakdown products of C3, C4, and properdin factor B in synovial fluids from patients with rheumatoid arthritis.

Synovial fluids from 31 patients with seropositive rheumatoid arthritis (RA) and 23 patients with seronegative RA had significantly increased levels of breakdown products to C3, C4, and properdin factor B when compared to patients with osteoarthritis (OA) (P less than 0.01). The same patients exhibited a considerable overlap of native C3, C4, and properdin factor B levels when their results were compared with those of OA patients. The parallel increase of C3d, C4d, and Ba levels in patients with RA suggests an activation of the complement system rather than a nonspecific enzymatic breakdown in synovial fluids.


Intracellular collagen fibers at the pannus-cartilage junction in rheumatoid arthritis.

Sections of the pannus-articular cartilage junction from a patient with rheumatoid arthritis (RA) were prepared for electron microscopy. Cells at this interface contained membrane-bound collagen fibrils, apparently in various stages of digestion. Previous studies have demonstrated intracellular collagen fibrils at sites of very active and rapid collagen resorption. It is suggested that at certain times in certain patients with RA, phagocytosis of cartilage collagen fragments and subsequent intracellular digestion may serve as a supplemental pathway to extracellular collagen degradation.


D-penicillamine in the treatment of rheumatoid arthritis.

Forty-four patients with definite or classic rheumatoid arthritis and failure to tolerate or respond to gold therapy were treated with D-penicillamine on a so-called go-slow, go-low regime. Seventeen patients tolerated the drug and had a 3-13 month follow-up assessment; 8 were markedly improved, 6 moderately or slightly improved, and 3 unimproved. Penicillamine had to be discontinued in 9 patients because of toxic side effects.


Activators and inhibitors of fibrinolysis in rheumatoid and nonrheumatoid synovial membranes. A histochemical study.

Histochemical studies of rheumatoid and nonrheumatoid synovial membranes revealed lowered fibrinolytic activity at sites of chronic inflammatory infiltrates and deposits of fibrin-like material (FLM). Inhibition of fibrinolysis occcurred over these areas and was always inversely related to the fibrinolytic activity. Hence the low fibrinolytic activity of these chronically inflamed tissues seems to result from the presence of inhibitors of fibrinolysis. Such inhibitors in FLM deposits may be responsible for the prolonged persistence of these deposits in the rheumatoid joint.


Enhancement of in vitro immunoglobulin synthesis of rheumatoid lymphocytes by aggregated human gamma globulin.

In vitro immunoglobullin synthesis by lymphocytes of patients with rheumatoid arthritis (RA) was enhanced by the addition of heat-aggregated human IgG. Stimulation occurred in the presence of fresh seropositive autologous plasma and was abolished by heat inactivation. By contrast, no significant stimulation was induced in lymphocytes from patients with miscellaneous arthritides and normal subjects cultured in the presence of either fresh autologous plasma or fresh normal AB serum. Although stimulation of RA lymphocytes did not occur in the presence of fresh AB serum, it did when purified IgM rheumatoid factor was added to this serum. These results suggest that immune complexes formed between aggregated IgG and rheumatoid factor in the presence of complement stimulate immunoglobulin synthesis in the peripheral blood lymphocytes of patients with RA.


Hyaluronic acid production by irradiated human synovial fibroblasts.

Radioactive particles as well as X-irradiation from an external source have been used in the treatment of rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In order to  clarify effects of ionizing irradiation on synovial cells, radioactive gold (198Au) and yttrium (90Y) were added to fibroblast cultures derived from human synovial membranes. Other cultures were irradiated by a Picker X-ray machine. Fibroblast growth and hyaluronic acid production were measured. Radioactive gold and yttrium particles induced a significant increase of hyaluronic acid synthesis rate (pg/cell/day) and inhibited fibroblast growth. Fibroblasts continued to overproduce hyaluronic acid and to show growth inhibition 3 weeds after irradiation with radioactive gold. Hydrocortisone inhibited hyaluronic acid overproduction induced by radioactive gold. Overproduction of hyaluronic acid induced by the X-ray machine was inhibited by hydrocortisone, actinomycin-D, and cycloheximide. Fibroblasts derived from normal and rheumatoid patients responded similarly to ionizing irradiation.


Differences in the collagen types synthesized by lapine articular chondrocytes in spinner and monolayer culture.

In five different secondary monolayer cultures of rabbit articular chondrocytes, 72-89% of the the collagen synthesized was Type 1, as determined by alpha chain separation and CNBr-cleavage peptide analysis. When sister cells were transferred to spinner bottles after primary monolayer culture growth, 88% of the collagen formed in four separate experiments was Type II. A reversion to Type I collagen synthesis occurred when the spinner-cultured cells were returned to monolayer flasks. The change in the species of collagen depended on the suspension condition and not on the low Ca2+ content (0.33 mM) of the spinner medium. These findings parallel the switch to phenotypic glycosaminoglycan synthesis that also occurs when monolayer-cultured cells are transferred to spinner bottles. Whether this phenomenon arises through a genetic control mechanism rather than environmental selection for particular cohorts of cells has not yet been determined.


Tendon calcifications in chondrocalcinosis. A clinical, radiologic, histologic, and crystallographic study.

Fine linear extraarticular calcium deposits were found in X-rays of 7 of 52 patients with articular chondrocalcinosis (ACC). Seven Achilles tendons, seven quadriceps tendons, and one plantar fascia were affected. In a control group of comparable age and sex, without ACC but with generalized osteoarthritis, no calcifications were found in the tendons. On a biopsy specimen of Achilles tendon with such calcium deposits, X-ray diffraction showed that they had the characteristics of calcium pyrophosphate dilhydrate. Isolated small foci of crystals were observed on some segments of tendon bundles. The presence of fine linear calcifications on X-rays of the Achilles or quadriceps tendons may be a useful aid in the radiologic diagnosis of so-called articular chondrocalcinosis.


Synovial chondromatosis affecting the temporomandibular joint. Case report and literature review.

A case of synovial chondromatosis affecting the temporomandibular joint is reported and the literature is reviewed. Chondromatosis occurs most frequently in this joint in middle-aged women, and it presents with pain and tenderness over the joint. Radiologically, the condition is detected by widening of the joint space with variable presence of radiodense loose bodies. Pathologically, the loose bodies in the case presented were demonstrated arise from cartilaginous metaplasia of the synovial lining. The stimulus for synovial chondrometaplasia at this site is unknown.


Chronic arthropathy associated with rubella vaccination.

Eleven children suffered recurrent ipisodes of knee stiffness ("catcher's crouch syndrome") after receiving HPV-77DK12 rubella vaccine. One was evaluated by arthroscopy and the synovium was found to be hypertrophied posteriorly and to protude in folds into the intercondylar notch and lateral joint space. Culture for rubella virus was unsuccessful. The author's observations provide evidence that "catcher's crouch syndrome" is a synovial disease and that chronic joint disease can be a sequela of rubella immunization.


Spontaneous rupture of the liver. An unusual complication of SLE.

A patient with systemic lupus erythematosus developed an acute surgical abdomen secondary to spontaneous rupture of the liver and died. Postmortem examination revealed an arteritis of the liver, pancreas, adrenal gland, skeletal muscle, and spleen. It appeared that an arteritis of the liver produced areas of infarction that ruptured and caused the clinical findings.


The university rheumatic disease clinic: provider and patient receptions of cost.

A medical school based rheumatic disease clinic provides patient services and educational activities and conducts clinical research. The patient population and routes of access to one such clinic are described, and the effect of direct and indirect costs influencing patient compliance are analyzed. It was found that the program cost of such a facility greatly exceeds the administrative cost. Alternatives to such high-cost facilitities are considered.


Effect of serum and liver extracts from hypercholesterolemic rats on the synthesis of collagen by isolated aortas and cultured aortic smooth muscle cells.

Rats were made hypercholesterolemic by feeding them a high-cholesterol, olive oil diet for one week. The effect of sera and 35,000 X g supernatants of liver homogenates on collagen synthesis was studied in isolated aortas, cultured arterial smooth muscle cells and the same cells in suspension. Compared to the preparations from normal rats, the liver preparations from hyperlipidemic rats stimulated collagen synthesis in both isolated aortas and cultured smooth muscle cells by about 25%. In these test systems hyperlipidemic serum was without effect but when added to smooth muscle cells incubated in suspension, produced a significant increase in the amount of collagen secreted. Hyperlipidemic serum caused an increase of about 50% in the incorporation of [3H]-thymidine by cultured smooth muscle cells.


Histogenetic differences between parent and daughter vessels of human coronary arteries.

The age-dependent changes of the parent vessels of human coronary arteries are characterized by a very early appearance and very intense development, as well as by a rapid association of degenerative processes. In contrast, the age-dependent changes of the daughter vessels of human coronary arteries are characterized by a very late occurrence and very slow development, as well as by the lack of associated degenerative processes. This histogenetic difference between parent and daughter vessels was not observed in other organ arteries. The two different histogenetic areas of the coronary tree appeared separated by a boundary constantly found at the level of the branching area, in a point where longitudinal muscle columns of the parent vessel criss-cross proximate to the branch orifice and describe half a spiral round it. No particular wall changes were observed when an epicardial branch became an intramyocardial one.


Tissue storage and control of cholesterol metabolism in man on high cholesterol diets.

The possibility of accumulation of tissue cholesterol in human beings submitted to high cholesterol feeding was investigated in liver biopsies and through fecal sterol balance studies. Feeding to 10 individuals 3.1 to 3.4 g/day of cholesterol for 3 weeks raised the mean serum level from 293 to 349 mg/100 ml, namely 19%, whereas the liver cholesterol content was 417 mg/100 g of wet weight. In 10 control cases eating 0.1--0.4 g/day of cholesterol serum cholesterol remained stable throughout the experimental period and the liver cholesterol content was 256 mg/100 g. Difference of liver colesterol level between the two groups was 62%. In 7 patients submitted to two periods of balance investigation on a cholesterol-free synthetic formula diet respectively prior to (PI) and after (PIII) eating the high cholesterol solid food from 4 to 15 weeks (PII), fecal steroid excretion in PIII exceeded PI in 3 patients. Such data are a direct evidence for the existence of an efficient system to release acutely stored cholesterol. In one patient bile acid excretion accounted for the difference between PIII and PI.


Dynamic mechanical properties of atherosclerotic aorta. A correlation between the cholesterol ester content and the viscoelastic properties of atherosclerotic aorta.

The effect of cholesterol and cholesterol ester content of aortas on the dynamic mechanical properties of these tissues was studied in rabbits during development of atherosclerosis. The disease was induced by feeding a 1.5% cholesterol diet for six weeks. At two week intervals, an equal number of control and experimental animals were sacrificed and their aortas were collected. After grading the aortas for lesions, one-half of each aorta was used for the determination of cholesterol and cholesterol ester content while the other half was used for the determination of viscoelastic properties. The viscoelastic properties were determined in both the longitudinal and the tangentail direction of the aortas. The results showed that cholesterol feeding had no effect on the longitudinal dynamic Young's modulus of the aortas. On the other hand, the tangential dynamic Young's modulus of the aortas was found to be very much influenced by the cholesterol diet. After the first two weeks of cholesterol feeding, the tangential dynamic Young's modulus was found to be increased markedly with time. At the end of the 6-week period, the average value of the tangential Young's moduli was 4.0 X 10(6) dynes/cm2 in the cholesterol-fed group while in the control group it remained 2.7 X 10(6) dynes/cm2. This increase in the tangential Young's moduli showed a remarkable correlation with the increase in the cholesterol ester content of the aortas. The cholesterol est content of aortas, like the tangential Young's modulus, remained unaffected during the first two weeks of cholesterol feeding. But after the first two weeks, a rapid increase in the cholesterol ester content was observed which coincided with an almost identical increase in the tangential Young's modulus values. It is concluded that during the early stages of atherosclerosis, the cholesterol ester content of aortas exerts a significant influence on the tangential dynamic Young's modulus of aortas. The serum levels of cholesterol and cholesterol ester, although increased steadily with the cholesterol feeding, did not show any direct correlation with the viscoelastic properties of the aortas.


The effect of cholestyramine on the faecal excretion of bile acids and neutral steroids in familial hypercholesterolaemia.

The faecal excretion of total bile acids was measured in two normal subjects and in seven patients with familial hypercholesterolaemia (four heterozygotes and three homozygotes) in the untreated state and during treatment with near-maximal doses of cholestyramine. There were no significant differences between the three groups. The increase in bile-acid excretion in response to cholestyramine was as great in the homozygotes as in the normal subjects. It is concluded that familial hypercholesterolaemia is not generally due to an inherited defect in the mechanisms for catabolizing cholesterol to bile acids.


A factor in yogurt which lowers cholesteremia in man.

Large dietary intakes of yogurt are found to lower cholestermia in man. This effect is associated with a reduction of incorporation of radioacetate into serum cholesterol. The effect appears slowly and persists after intake of the yogurt stops suggesting that the mechanism involves the synthesis of a regulatory protein rather than an allosteric effect. The effective agent is postulated to be hydroxymethyl glutarate which inhibits the regulatory enzyme hydroxymethyl glutaryl CoA reductase (EC 1.1.1.3.4).


Enhanced synthesis and accumulation of collagen in cholesterol-aggravated pigeon atherosclerosis.

Atherosclerotic segments of pigeon aorta synthesized collagen at four times the rate found in normal aorta (Athero = 2071 +/- 1339 ng/g/h; Control = 497 +/- 192 ng/g/h; P less than 0.025). Similar results were obtained when synthesis was expressed per mg DNA. Elevation in collagen synthesis was relatively specific, collagen accounting for 4% of total protein synthesis in lesion-free aorta and 11.5% in atherosclerotic aorta. Substantial increases in total collagen were observed in atherosclerotic aortas (Athero = 9.9 +/- 3.1 mg/aorta; Control = 6.0 +/- 1.3 mg/aorta; P less than 0.05). Ultrastructural studies revealed the accumulation of large amounts of dense fibrillar collagen in the sub-endothelial region of the plaque. Plaque cells contained multiple vacuoles, an extensive rought endoplasmic reticulum and many mitochondria, suggesting active protein synthesis. It is concluded that increased collagen biosynthesis and deposition is an important metabolic derangement in lipid-rich atherosclerotic lesions whihc promotes their gradual conversion to fibrous plaques.


Tissue lipoprotein lipase in the hyperthyroid rat. Effect of growth and aging.

The hydrolysis of lipoprotein triglycerides is catalyzed by lipoprotein lipase (LPL), an enzyme found in many tissues. We have examined tissue LPL activity (LPLA) in rats with experimentally-induced hyperthyroidism. In younger, lighter rats, hyperthyroidism was accompanied by a decrease in LPL in adipose tissue whereas heart and diaphragm muscle LPL activities were increased. These changes are consistent with the hypothesis that the hypercatabolism and increased beta-adrenergic activity of hyperthyroidism result in characteristic changes in tissue LPLA. In older, heavier hyperthyroid animals, however, adipose tissue LPLA was increased and heart and diaphragmatic LPLA were similar to control activities. Propranolol feeding abolished the thyroxine-induced increase in adipose tissue LPLA. In euthyroid animals of similar size the response of muscle LPLA to short-term starvation was also attenuated. These changes in tissue LPLA may provide a mechanism for shunting triglyceride fatty acids away from adipose tissue for utilization by muscle in the hyperthyroid state. During growth and aging, these adaptations are modified.


A factor in milk which influences cholestermia in rats.

Rats fed cholesterol show an adaptive response over several weeks with lowering of the initial rise of choleseremia to near normal levels. This reducation of cholesteremia is augmented by including milk powder in the diet. A similar effect is obtained by feeding 0.1% beta-hydroxy-beta-methylglutaric acid (HMG) in the diet. The evidence suggests that milk powder contains HMG.


Endomyocardial fibrosis and eosinophilia.

Absolute eosinophil counts were assessed in 15 African patients with proven endomyocardial fibrosis. Though the mean eosinophil count in patients with endomyocardial fibrosis was higher compared with the normals reported from Kampala (1-13 vs 0.72X10(9)/1), the absolute range was comparable. A high percentage of patients with endomyocardial fibrosis had malarial parasites, high malarial antibody titres, hookworms, or strongyloides, but the correlation of eosinophilia to various parasitic infections was poor. Both eosinophilia and parasitic infections are common in the tropics and they effect patients with endomyocardial fibrosis no more than the population at large. Other aetiological factors, genetic, environmental, and immunological, are felt to be important in the causation of endomyocardial fibrosis in Uganda and evidence for this is reviewed. Though there is a similarity in pathological features, African endomyocardial fibrosis is a distinct entity from Löffler's endocarditis and cardiac lesions seen in eosinophilic leukaemia or reactive eosinophilia. There is no hard evidence to suggest that African endomyocardial fibrosis is a variant of Löffler's endocarditis caused by parasitic infections via eosinophilia.


A study of systolic time intervals during uninterrupted exercise.

A method has been developed for obtaining measurements of the systolic time intervals during uninterrupted graded exercise in the upright position on a bicycle ergometer. The method has been applied to 112 subjects divided into 4 groups: two of normal subjects below and above the age of 40, respectively (mean ages 30 and 48 years), and two of patients (mean ages for each about 50 years), both with coronary insufficiency, but one without and the other with abnormality of left ventricular function as shown by ventriculography. The measurements obtained yield linear relations between total electromechanical systole (QS2) and heart rate, between pre-ejection period (PEP) and RR interval for each subject. The average standard deviation about the regression is less than 6 ms for all three regression lines; the average correlation coefficient is greater than 0-93. The younger group of normal subjects have a significantly shorter PEP compared to the older group. Indices have been derived which separate the patient groups from each other and from the normal subjects. Using these indices 86 per cent of all the subjects were correctly classified according to the group to which they belonged. It is concluded that measurements of STI during uninterrupted exercise offer valuable information in the assessment of cardiac patients.


Changes in systolic time intervals during treatment of diabetes mellitus.

The cardiovascular response to the control of diabetes by sulphonylurea drugs has been investigated using systolic time intervals in a group of 19 diabetics. Before treatment a significantly greater heart rate and shortening of QS2 interval and left ventricular ejection time index were encountered among the more hyperglycaemic diabetic patients requiring drug therapy while all diabetics had a reduction of pre-ejection period index. There was a gradual return of resting heart rate and systolic time intervals to control values along with the fall in plasma sugar concentration during treatment. A likely explanation of the findings is that uncontrolled diabetics, particularly those more severely affected, are subjected to an increased adrenergic stimulus to the cardiovascular system which disappears with therapy. There is no evidence of positive inotropic action of sulphonylurea drugs in this study.


Echocardiographic evaluation of left-to-right shunt in ventricular septal defect and persistent ductus arteriosus.

Forty-five patients with either a ventricular septal defect or a persistent ductus arteriosus were assessed by echocardiography and cardiac catheterisation. Left atrial dimension was expressed either as a function of the body surface area (LAD cm per m2 BSA), or as a multiple of the aortic root dimension (LAD/AR), and was compared with the shunt size as determined by oximetry. A highly significant (P less than 0-001) regression relation was found for the group as a whole. A significant relation also existed for each individual group: ventricular septal defect, ventricular septal defect with pulmonary hypertension, and persistent ductus arteriosus. Regression equations were derived for the whole group. The value of echocardiography is in separating large from small shunts and in adding a dimension to the follow-up of the individual patient.


Echocardiographic diagnosis of primitive ventricle with two atrioventricular valves.

Anatomical studies have shown that the pathognomonic feature of primitive ventricle is absence of that portion of the interventricular septum which interposes between the atrioventricular valves. The relation of this posterior septum to the atrioventricular valves is such that echocardiography should be a particularly suitable technique for showing its presence or absence. When a posterior septum is present, it is seen on an echocardiogram as a double echo between the two atrioventricular valves. It presence limits posterior movement of the septal cusp of the anterior atrioventricular valve. Absence of the posterior septum should, therefore, be seen echocardiographically as more than mere absence of the double septal echo. It should be possible to show unusual posterior excursion of the "septal" cusp of the anterior atrioventricular valve and apposition of the "septal" cusps of the atrioventricular valves during diastole. In most instances it should also be possible to demonstrate that the posterior great artery is in continuity with both atrioventricular valves. We have shown these echocardiographic features in 26 patients. The diagnosis of primitive ventricle has been confirmed at necropsy or at operation in 4 patients. In the other 22 patients in angiographic data are compatible with a diagnosis of primitive ventricle. Demonstration of these positive features at echocardiography is, therefore, of considerable value in the initial diagnosis of primitive ventricle and in particular its differentiation from other congenital malformations.


Natural history of the ventricular septal defect in tricuspid atresia and its surgical implications.

We have previously reported isolated cases of anatomical and functional closure of ventricular septal defects in tricuspid atresia. To study this phenomenon further, clinical, angiographic, and pathological findings in 20 consecutive cases of tricuspid atresia were reviewed. Sixteen cases were found to have normally related great arteries (type I) and 4 had transposition (type II). In 6 of these patients there was evidence of closure of a ventricular septal defect; in 3 this was complete and in the other 3, partial. Five of these ventricular septal defect closures occurred in type I patients and one among type II. Increasing cyanosis and polycythaemia and/or disappearance of a previously heard murmur were observed in all patients; these signs are more conspicuous in complete closure than in partial. The incidence of closure of ventricular septal defect in tricuspid atresia is 38 per cent and approximates to the incidence of spontaneous closure of isolated ventricular septal defects. Progressive muscular "encroachment" of the margins of ventricular septal defects with subsequent fibrosis and covering by endocardial proliferation is the most likely mechanism of closure in tricuspid atresia. The factors initiating the closure of ventricular septal defect remain unknown, but because of its occurrence in patients both with and without previous shunt operations, it is reasonable to assume that closure of ventricular septal defect in tricuspid atresia is not initiated or accelerated by these surgical shunts. Recent developments in surgical technique permit total surgical correction of tricuspid atresia at least in a physiological sense, but these operations can be successfully performed only in older children (over 8 years). Thus, palliation is essential in younger patients until they reach the age for total correction. A Blalock-Taussig shunt is recommended in preference to a Glenn anastomosis because the latter may leave the left pulmonary circuit without blood supply if the ventricular septal defect closes. In type II cases, a large and non-restrictive ventricular septal defect is essential for survival of the patient after the Fontan operation. For this reason, the size of the ventricular septal defect should be evaluated before and at the time of surgical correction. If the ventricular septal defect is small in a type II case, resection of the ventricular septum or a complete bypass of the ventricular septal defect and right ventricle is necessary at the time of the Fontan operation.


Coronary artery occlusion and alcohol intake.

A group of over 900 patients who underwent coronary arteriography were asked about their alcohol intake. The patients who were abstainers or consumed less than an equivalent of 180 ml absolute alcohol per week had higher coronary artery occlusion scores than the group consuming more than that amount, though the latter group smoked more and had higher plasma triglyceride levels.


Rheumatic aortic stenosis in young patients presenting with combined aortic and mitral stenosis.

This report describes 30 patients under the age of 30 years with rheumatic aortic stenosis, presenting with combined aortic and mitral stenosis. Three patients had additional tricuspid stenosis. Twenty-eight patients gave a history of rheumatic polyarthritis. The diagnosis was confirmed by right and left heart catheterisation in all. The murmur of aortic stenosis was not initially present in 8 out of 10 patients in congestive heart failure. Aortic valve calcification was not seen. Cineangiography showed a tricuspid aortic valve in all, unlike congenital aortic stenosis. A unique feature of this group was the raised pulmonary vascular resistance in 87 per cent of the patients. The present study shows that patients in India developing aortic stenosis after rheumatic fever do so early in the natural history of the disease.


Left juxtaposition of the auricles with l-position of the aorta. Report of 6 cases.

Six hearts are described in which juxtaposition of the auricles to the left of the great arteries (left juxtaposition) was associated with l-postion of the aorta. Atrial anatomy was abnormal but constant; the os of the right auricle occupied the expected site of the atrial septum, producing posterior and inferior desplacement of this structure. This is of importance since the os could be mistaken for a defect of the atrial septum, while the displacement of actual defects is of significance to any intended catheterisation procedure including atrial septostomy. Five of the hearts showed atrioventricular concordance while the other specimen was an example of primitive ventricle with a right-sided outlet chamber. The ventriculoarterial connection in this latter case was normal, with l-malposition (anatomically corrected malposition). Ventriculoarterial connections in the other hearts were transposition in 3, single aortic trunk with pulmonary atresia in 1, and an intermediate between double outlet right ventricle and transposition in the other. These cases confirm the observation that juxtaposition of the auricles is associated with severe cyanotic congenital heart disease.


Clinical and electrophysiological effects of intravenous quinidine in man.

Quinidine gluconate (total dose 4-4 to 9-1 mg/kg) was infused intravenously over 22 minutes in 20 patients with either frequent premature ventricular contractions or supraventricular arrhythmias, 16 of whom had bundle-branch block. Therapeutic plasma quinidine levels (3 to 7 mg/l) were achieved in 15. Heart rate, atrioventricular nodal, and infranodal conduction times did not change significantly. The QRS duration increased significantly from 128+/-30 to 134+/-29 ms at peak plasma quinidine levels (P less than 0.01). Mild hypotension occurred during infusion in most patients. Two patients had a severe but transient toxic response characterised by hypotension, nausea, vomiting, and diaphoresis. Atrioventricular dissociation with escape His bundle or fascicular rhythm occurred in 1 patient with sinus bradycardia. Bundle-branch block does not contraindicate administration of quinidine. Quinidine gluconate administered intravenously (0-3 to 0-4 mg/kg per min) is frequently associated with hypotenstion and should be used only in an intensive care setting and with careful monitoring of blood pressure.


Effect of positive acceleration (+gz) on electrocardiogram of subjects with vasoregulatory abnormality.

ST-T wave changes in the electrocardiogram detected during routine examination and aggravated by erect posture, hyperventilation, and exercise in apparently healthy young individuals have been termed vasoregulatory abnormalities. No evidence of ischaemic heart disease has been found in such subjects. Ten young healthy air crew with vasoregulatory abnormalities were subjected to maximal exercise on treadmill and procedure repeated after 120 mg propranolol daily for 3 days. After one week, they were subjected to a stress of positive acceleration (+gz) in a human centrifuge at 2-5 g and 3-5 g for 15 seconds each at a constant rate of rise of 0-1 g/s and the electrocardiogram was monitored during and in the post-acceleration phase. The procedure was repeated after propranolol 120 mg daily for 3 days. The stress of positive acceleration resulted in pronounced prominence of P waves and inversion of T waves (as has been reported in normal subjects) with minimal ST depression in the electrocardiogram. ST segment depression during exercise, at heart rates corresponding to those achieved during peak centrifuge runs, was significantly more pronounced. The ST, P, and T wave changes were returned to normal after propranolol. It is concluded that minimal ST segment depression after stress of positive acceleration as compared with conspicuous ST segment depression during exercise at corresponding heart rates, and their normalisation after propranolol, rules out ischaemia as an aetiological factor in subjects with vasoregulatory abnormalities.


Right-sided endocarditis involving both tricuspid and pulmonary valves in a patient with ventricular septal defect.

A patient with alpha-haemolytic streptococcus endocarditis on a ventricular septal defect is described. The disease spread to involve both tricuspid and pulmonary valves which were largely destroyed. The patient developed severe right heart failure with pronounced rise in right atrial pressure. This led to right-to-left shunting through the foramen ovale with systemic embolization. She was successfully treated by operative closure of the defects and double right-sided valve replacement.


Pulmonary embolisation of retained transvenous pacemaker electrode.

When removal of an endocardial pacemaker electrode became necessary in a patient with congestive myocardiopathy, it proved to be incarcerated. Therefore, a portion of it had to be left in site after cutting the external end of the pacing catheter. During follow-up examinations the electrode was seen to migrate in the large veins and finally was found embolized into a pulmonary artery. While there have been publications on the migration of retained pacemaker electrodes before, to our knowledge this represents the first case reported with pulmonary embolization of the migrating fragment.


Wolff-Parkinson-White syndrome. Circus movement tachycardia dependent on procainamide.

A patient with Wolff-Parkinson-White syndrome was disabled by rapid ventricular rates during atrial arrhythmia and by periods of asystole. Circus tachycardia was induced by atrial stimulation only after the administration of procainamide. Nevertheless, treatment with this drug controlled the ventricular rate and spontaneous circus tachycardia did not occur.


Whole-body albumin mass and distribution in rats fed on low-protein diets.

1. From 5 weeks of age, control and experimental rats were given diets containing 210 and 31 g protein/kg respectively, and killed for analysis at 0, 2, 5, 8, 12 and 20 d after the start of the experiment. At these times estimates were made of plasma albumin concentration, plasma volume and total vascular and extravascular albumin mass. 2. Plasma albumin concentrations were significantly lower in the experimental animals when compared to controls at 8, 12 and 20 d but plasma volumes (ml/kg body-weight) tended to be greater in the former animals. Total vascular albumin mass (g/kg body-weight) was significantly less in experimental animals compared to controls at 8 and 20 d, but was significantly reduced below values at 0 d only at 20 d. 3. extravascular albumin mass (g/kg body-weight) was significantly lower in experimental animals in comparison with controls at 2, 5, 8, 12 and 20 d and significantly reduced below values at 0 d at 5, 8, 12, and 20 d. 4. Whole-body albumin mass was significantly reduced at 5, 8, 12 and 20 d when compared both with controls killed at the same time and animals killed at 0 d. Measurement of the ratio, extravascular albumin mass: vascular albumin mass indicated a significant redistribution of whole-body albumin mass at 5 and 20 d and mean values for this ratio were always lower in experimental animals than in controls. 5. It was concluded that measurement of plasma albumin concentration does not indicate the true extent of whole-body albumin losses in protein deficiency since total vascular albumin mass is, to some extent, maintained at the expense of extravascular albumin mass.


The effect of feeding magnesium-enriched diets on the quality of the albumen of stored eggs.

1. Pullets were given from 1-d-old diets containing 1-6, 4-1, 8-1 and 12-0 g Mg/kg. Only small effects of these diets on live weight, food consumption, egg number, egg weights or egg-shell thickness were observed except at the highest level (12-0 Mg/kg) which caused diarrhoea and an appreciable lowering of the live weight of growing pullets. A further group was given from point-of-lay a diet containing 9-3 g Mg/kg. 2. Eggs laid on 3 consecutive days from each of eighteen hens were collected at intervals of 3 weeks until the birds were 68-5 weeks old. Eggs laid on the 3rd day were used to determine the initial proportion of thick egg-white present and also the concentration of Mg, Ca, Na and K in the thick egg-white. Eggs laid on the 1st and 2nd days were stored at 20 degrees for 20 d to establish the proportion of thick egg-white remaining after storage. 3. With the unsupplemented diet the proportion of residual thick egg-white after storage of eggs for 20 d at 20 degrees was 306, 161 and 305 mg/g total egg-white when the hens were 26-5, 53-5 and 68-5 weeks of age respectively. When the diet containing 9-3 g Mg/kg was given, the proportion of thick egg-white after storage remained approximately 400 mg/g throughout the period of the trial. 4. The mean Mg concentration in the thick egg-white of eggs laid by hens given unsupplemented diets was 5-77 mM. The addition of extra Mg to the diet increased the content of Mg in the thick egg-white, for example when the diet contained 9-3 g Mg/kg the mean concentration rose to 7-69 mM.


Intravenous infusion of glucose and insulin in relation to milk secretion in the sow.

1. A comparison was made of the composition of milk from front and rear tetas in four sows. There were small and not significant differences in fat, protein and lactose contents, and in the fatty-acid composition of the milk fat with the exception of the 18:3 acid where the difference was also small but significant. 2. The effects of intravenous infusions of glucose and insulin in lactating sows on milk secretion and blood composition were investigated in two sows. 3. Intravenous infusion of glucose had no effect on blood plasma glucose concentration but increased the yields of lactose, protein and water. 4. Intravenous infusion of insulin depressed plasma glucose concentration and the yields of lactose and water. The yield of protein was unaffected. 5. It is concluded that differences between the non-ruminant (the sow) and the ruminant in the responses in milk secretion to glucose infusion may be related to differences in the sensitivity to insulin of mammary tissue.


Some effects of variation in carbohydrate and nitrogen intakes on the chemical composition of mixed rumen bacteria from young steers.

1. Samples of rumen digesta were taken at different times after giving calves various dried forage or hay and cereal diets and mixed bacteria were separated. 2. For calves receiving dried forage only, the carbohydrate content of mixed bacteria varied with time after feeding, reaching a maximum of approximately 140 g/kg dry matter (DM) after 0-5-1h, decreasing to about 60 g/kg DM after 4 h. 3. Replacement of part of the dried forage with glucose to give a similar metabolizable energy intake but approximately half as much nitrogen, produced a similar pattern of change with time after feeding but resulted in a markedly increased maximum bacterial carbohydrate content (approximately 230 g/kg DM). Addition of urea to this diet reduced the bacterial accumulation of carbohydrate to approximately the same level as was found in samples from calves receiving forage only diets. For the forage diets the carbohydrate content fell to 60-100 g/kg DM 4 h after feeding. For the hay and cereal diet the value was 170 g/kg DM at this time. 4. Changes in bacterial carbohydrate content were largely a result of changes in storage polysaccharide (alpha-dextran) content. 5. Crude protein (N X 6-25) and ash contents of mixed bacteria decreased after feeding with all-forage diets, but returned to approximately fasting levels within 4 h. At this time samples from calves receiving dried forage and glucose diets contained less CP and more ash (approximately 450 and 220 g/kg DM respectively) than those from calves given diets of dried forage only (approximately 525 and 180 g/kg DM respectively). 6. CP, total carbohydrate and ash content of mixed rumen bacteria contributed approximately 0-80 g/g DM. 7. Sodium, potassium, calcium, magnesium and phosphate accounted for approximately 0-50 g/g bacterial ash.


Methionine sulphoxide as a source of sulphur-containing amino acids for the young rat.

1. Young male rats were used in five experiments to study the utilization for growth of methionine sulphoxide, and the relationship between the sulphoxide content in the diet and the level of microbiologically determined methionine activity in blood or blood plasma. In one nitrogen-balance experiment methionine and methionine sulphoxide were compared as supplements to a casein diet and a fish-meal diet. 2. Methionine sulphoxide was poorly utilized for growth when testd as the sole sulphur amino acid in an amino acid diet. Substitution of one-third of the sulphoxide with cystine improved utilization so that it approached that of methionine. 3. Methionine alone and in combination with methionine sulphoxide were added to a soya-bean-meal diet. The sulphoxide showed no adverse effect on growth. 4. Fish meal in which methionine had been oxidized to methionine sulphoxide was tested alone and in combinations with unoxidized fish meal. Only when the oxidized meal was given alone was there an appreciable effect on growth. The fish meal used were low in cystine. 5. Whereas both methionine and methionine sulphoxide improved the N balance when a casein diet was given, there was no effect when a fish-meal diet was given. 6. There was a linear relationship between methionine sulphoxide content in the amino acid diets and the methionine activity in the blood plasma. Methionine sulphoxide added to a soya-bean-meal diet or present in oxidized fish meal gave a curvilinear relationship, and the observed activities were lower than with the amino acid diets. Methionine activity in blood could not be used as an indicator of moderate amounts of methionine sulphoxide in protein-containing diets.


Polyadenylic-polyxanthylic-polyuridylic acid triple helix.

Poly(xanthylic acid) [X)n] abolished the ability of (A)n-(U)n to induce interferon in "superinduced" (actinomycin D and cycloheximide) primary rabbit kidney cells. Under the same conditions, (X)n had a relatively minor effect on the interferon inducing capacity of (I)n-(C)n. Evidence based on mixing curves, melting profiles, pancreatic ribonuclease resistance and sucrose gradient ultracentrifugation pointed to the conclusion that the following three reactions occur depending on stoichiometry: (1) 2(A)n-(U)n + (X)n leads to (A)n-2(U)n + (A)n-(X)n; (2) (A)n-(U)n + (X)n leads to (A)n-(U)n-(X)n; (3) (A)n-(U)n + 2(X)n + (X)n-(U)n. The second reaction represents the formation of a new triple helix which can also be formed according to the following reactions: (X)n-(U)n + (A)n leads to (A)n-(X)n-(U)n; (A)n-(X)n + (U)n leads to (A)n-(X)n-(U)n.


Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 1. Dinucleoside monophosphates.

The base components of underivatized oligodeoxynucleotides can be determined qualitatively by mass-spectral analysis at the nanogram level. The thermal and electron-impact conditions of the spectrometer allow the cleavage of the phosphodiester bonds of the oligonucleotide chain, resulting in fragments (I), whose mass identifies the base, and other fragments (II), which contain the purine or pyrimidine base plus portions of the deoxyribose and the phosphate moieties. A study of 16 dinucleoside monophosphates indicates that the relative intensities of the m/e values of the type II fragments are significantly and reproducibly different for sequence isomers. From the complex spectra of dinucleoside monophosphates, specific ions for each mono-nucleotide residue have been selected which reveal the location (5' or 3' terminus) of the bases in the dinucleoside monophosphate. These ions appear in spectra of deoxyribonucleic acid fragments as well as in model compounds. A simple computer program has been devised which utilizes ion ratio values to determine sequence. The method is applicable to oligonucleotides of longer length.


Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 2. Application of computerized pattern recognition to sequence determination of di-, tri-, and tetranucleotides.

A novel strategy for the sequence analysis of oligodeoxyribonucleotides has been devised which is based upon the analysis of intact underivatized oligonucleotides by mass spectrometry followed by interpretation of the mass-spectral data by computerized pattern-recognition techniques. The pyrolytic and electron-impact conditions of the mass spectrometer permit the cleavage of oligonucleotides of varying chain length and composition, yielding reproducible fragmentations and characteristic m/e values which can be used to reveal purine and/or pyrimidine base sequence information. The selection of optimum features (which are the ratios of peak heights of specific ions, or the linear combination of such ratios) has been done by an interactive feature selection method employing multidimensional k nearest-neighbor analysis and two-dimensional feature-space plots (nonlinear mappings) of the mass-spectral data. Features have been found which allow 100% classification accuracy in predicting the 5' and 3' terminus of all of the dinucleotides commonly found in DNA. Other specific features have been found which indicate adjacent nucleotides within a tetranucleotide. Knowledge of the adjacent nucleotide pairs present, in conjunction with the information as to the 3' or 5' position of the residues in each pair, permits the reconstruction of the sequence of the tetranucleotide.


Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA.

The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.


Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 2. Amino acid sequence of labeled peptides.

The purification and characterization of two related peptides making up the glutamine binding site of formylglycinamide ribonucleotide amidotransferase from chicken liver have been presented. An amino acid residue(s) involved in binding glutamine to the enzyme was selectively labeled with [14C]iodoacetate. The labeled enzyme was reduced, carboxymethylated, and degraded by trypsin to a large radioactive peptide that yielded on acid hydrolysis only cysteine as a radioactive carboxymethylated derivative. The tryptic peptide was further digested with a protease from Streptomyces griseus. Two radioactive fractions (I and II) were obtained by gel filtration on Sephadex G-25. Furthermore, two 14C-containing peptides have been isolated from fraction I by the aid of ion exchange chromatography on AG 1-X2, AG 50W-X2 and DEAE-cellulose. Upon acid hydrolysis both peptides yielded only carboxymethylcysteine (CMCys), cystine, glycine, valine, aspartic acid, and glutamic acid. The partial sequences of the amino residues in these peptides, which are located at the glutamine binding site, have been established by the dansyl-Edman method. The sequences of amino acids of peptides a and b are Gly-Val-Cys([14C]CM)-Asp-Asx-Cys(CM)-Glx...and Gly-Val-Cys([14C]CM)-Asx-Asx..., respectively. The two peptides are undoubtedly derived from the same segment of the original protein.


Stereochemistry of methylene transfer involving 5,10-methylenetetrahydrofolate.

The stereochemistry of the transfer catalyzed by rabbit liver serine transhydroxymethylase (EC 2.1.2.1) of the prochiral hydroxymethyl group from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate was studied. Initial kinetic studies on labeled 5,10-methylenetetrahydrofolate showed that nonenzymatic racemization of the prochiral methylene center was buffer dependent and was slow under the conditions employed. Specifically tritiated (3R)- and (3S)-serines were employed to study the transfer reaction. Reactions were carried out under various conditions and the stereochemistry of the methylene carbon of the 5,10-methylenetetrahydrofolate produced was determined. The enzyme was shown to be partially stereospecific for this transfer reaction, proceeding with a loss of about 50% of the stereochemical purity of the transferred carbon center. Possible mechanistic interpretations of this finding are discussed.


Mandelate racemase from Pseudomonas putida. Absence of detectable intermolecular proton transfer accompanying racemization.

An equimolar mixture of DL-[alpha-2H]- and DL-[alpha-13C]mandelate, when incubated with mandelate racemase (EC 5.1.2.2), shows conversion of singly labeled mandelate to unlabeled mandelate, due to solvent exchange of the alpha proton, while the level of doubly labeled mandelate remains at a constant low level. Similarly, an equimolar mixture of unlabeled and DL-[alpha-2H,alpha-13C]mandelate, when incubated with the enzyme, shows conversion of doubly labeled mandelate to singly labeled mandelate, due to solvent exchange, while the level of unlabeled mandelate remains constant at 50%. Incubation of an equimolar mixture of DL-[alpha-3H]mandelate and DL-rho-chloromandelate. These results indicate that mandelate racemase does not catalyze an intermolecular proton transfer to achieve racemization. These data are necessary, but not sufficient, results to indicate that mandelate racemase operates via a one-acceptor mechanism, in which the proton abstracted from one stereochemical face of a substrate molecule is returned to the opposite face of the same carbon of the substrate molecule.


Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure.

Nuclear magnetic resonance (NMR) spectroscopy of the exchangeable protons, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O has been found to be a powerful method to study the active site of the enzyme. This technique has been employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that zinc alone organizes the active site structure. All eight histidines per subunit of apoenzyme react with DEP. The accessibility of these residues to solvent is borne out by the broad, featureless NMR spectrum of the apoprotein. In the holoenzyme only His-19, which is exposed to solvent, can be modified with DEP. The reduced holoenzyme shows a well-resolved NMR spectrum compared with the oxidized form in which the lines are broadened by the paramagnetic copper ion. A spectrum very similar to that of the reduced enzyme is generated by addition of one zinc ion per subunit of apoprotein showing that zinc alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of zinc per subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios greater than or equal to 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.


Comparison of the base-sequence complexities of polysomal and nuclear RNAs in growing Friend erythroleukemia cells.

The base-sequence complexities of polysomal poly(A)+ RNA, nuclear poly(A)+ RNA, and total nuclear RNA from Friend erythroleukemia cells in logarithmic phase of growth were determined by measuring the proportion of labeled unique mouse DNA sequences which formed hybrids when incubated with a vast excess of each RNA. It was estimated that the RNAs had been transcribed from 1.8, 7.6, and 8.3%, respectively, of the haploid mouse genome. Although these estimates for polysomal and nuclear poly(A)+ RNAs were 2.5 times greater than those previously determined by analysis of the kinetics of the hybridization reactions between the RNAs and the cDNAs transcribed from them, they confirmed that the base-sequence complexity of nuclear poly(A)+ RNA in these cells is at least four times greater than that of polysomal poly(A)+ RNA.


Conformation of aromatic-substituted dinucleoside monophosphates: an extension of the base-displacement theory of carcinogenesis.

The conformations of 12 dinucleoside monophosphates containing N4-phenylcytidine (CPh) or N4(beta-naphthyl)cytidine (CbetaN) residues have been studied, using circular dichroic spectroscopy. The following compounds had aqueous spectra resembling their parent compounds, which lacked the modifying aromatic substituent: CPhpU, CbetaNpU, UpCPh, UpCbetaN, CPhpG, CbetaNpG, GpCPh, and CPhpA. The spectra of GpCbetaN and ApCbetaN were almost the reverse of the unmodified compounds, while CbetaNpA and ApCPh were intermediary in character. When the spectra were run in methanol, all major differences between the modified and unmodified compounds disappeared. This result suggested that the differences observed in aqueous solution were the result of stacking interactions between the aromatic ring and a neighboring purine. When the aromatic ring was naphthalene, the modified cytidine occupied the 3'-terminal position, and, when the purine was adenine, the effect was enhanced. These conclusions were supported by a consideration of chemical shifts in the 1H NMR spectra of ApCbetaN, and GpCbetaN, as compared to those of the unmodified compounds and CbetaNpG. A study of molecular models of ApCbetaN and GpCbetaN revealed a unique conformation in which the purine rotates to a syn position, in order to allow a close stacking interaction with the naphthalene ring. No such conformation is available for CbetaNpA and CbetaNpG, and the best partial stacking interaction occurs in a conformation with the purine in the anti conformation. The base-displacement theory of carcinogenesis (Levine, A. F., Fink, L. M., Weinstein, I. B., and Grunberger, D. (1974), Cancer Res. 34, 319) describes the conformational change resulting from the attachment of a bulky aromatic residue at the 8 position of guanine in RNA or DNA, and attributes biological importance to the event. The changes that occur upon substitution of the amino group of cytosine differ in detail from the above, but would be expected to produce similar biological results. Base-displacement effects need not be limited, therefore, to a particular substitution position in a nucleic acid.


Histones H2a, H2b, H3, and H4 are present in equimolar amounts in chick erythroblasts.

A quantitative labeling method for stoichiometric analysis has been applied to an investigation of the histone composition of chick erythroblast chromatin. The necessity of long fractionation procedures and the dangers of preferential extraction and staining are eliminated through the use of this simple procedure which involves the direct analysis of nuclear proteins on sodium dodecyl sulfate gels. Only a knowledge of the amino acid compositions of the histones is required. H2a, H2b, H3, and H4 are found to be present in nearly equimolar quantities.


Synthesis and function of 9,12,15-octadecatrien-6-ynoic acid in the moss ceratodon purpureus.

Biosynthesis of all-cis-9,12,15-octadecatrien-6-ynoic acid in the moss, Ceratodon purpureus, was studied using protonemata cultures with labeled 9,12,15-octadecatrienoic (linolenic) and 6,9,12,15-octadecatetraenoic acids as substrates. Both acids were efficiently converted into the acetylenic and into 5,8,11,14,17-eicosapentaenoic acids. Accordingly, the introduction of a triple bond in position 6 of linolenic acid involves formation of a double bond as a discrete step. Acetylenic acid triglycerides are reserve lipids in the moss. Under suitable growth conditions the acetylenic acids are catabolized and partly reused via acetate for de novo synthesis of fatty acids. They are not used for more direct syntheses of the common polyunsaturated fatty acids.


Transferrin receptor of the rabbit reticulocyte.

A Triton X-100 solubilized macromolecular complex of transferrin and a membrane constituent can be isolated by gel chromatography from rabbit reticulocytes previously incubated with 125I-labeled transferrin. The apparent molecular weight of this complex is close to that of ferritin, or about 445 000. On sodium dodecyl sulfate gel electrophoresis the complex displays two glycoprotein subunits, of molecular weights 176 000 and 95 000 in addition to transferrin. A transferrin-binding fraction with a molecular weight near 400 000, containing these subunits, can also be identified in membranes of nonincubated reticulocytes. The corresponding membrane fraction from mature erythrocytes, which have lost transferrin-binding activity, displays both protein subunits, but the 176 000 molecular weight component fails to give a PAS stain for carbohydrate. Treatment of reticulocytes with Pronase, which destroys the ability of the cells to form specific complexes with transferrin, degrades both components. We believe these results are consistent with the hypothesis that the primary transferrin receptor of the rabbit reticulocyte is a glycoprotein of molecular weight in the range 350 000-400 000, comprised of a combination of two subunits with molecular weights 176 000 and 95 000, respectively. Transferrin-binding activity appears to depend on the carbohydrate moiety of the 176 000 subunit.


Photoinduced size change of membrane vesicles from bovine retinal rod outer segment.

Quasielastic light-scattering measurements were made on isolated, osmotically swollen, bovine rod outer segment (ROS) disk membranes in aqueous Ficoll. The translational diffusion coefficients determined were (1.66 +/- 0.21) X 10(-9) and (2.31 +/- 0.14) X 10(-9) cm2/s at 25 degrees C for the unbleached and bleached membranes in 5% Ficoll, respectively, yielding the Stokes radii of 0.53 +/- 0.07 micronm for the former and 0.38 +/- 0.02 micronm for the latter. This photoinduced size decrease, which corresponds to a 60% decrease in the intravesicular volume, is far more pronounced than thos reported by others for ROS fragments.


Effect of lysolecithin on the structure and permeability of lecithin bilayer vesicles.

In order to elucidate the role of lysolecithin in membranes, we have examined the effect of lysolecithin on the structure and permeability of lecithin bilayer membranes. Small L-alpha-dimyristoyllecithin (DML) vesicles with myristoyllysolecithin (MLL) incorporated as well as small L-alpha-dipalmitoyllecithin (DPL) vesicles with palmitoyllysolecithin (PLL) were studied by nuclear magnetic resonance (NMR) methods at temperatures both above and below the alpha-gel in equilibrium liquid crystalline phase transition temperature (Tc) and as a function of the concentration of the incorporated lysolecithin. Europium (III) ion was used as a probe to measure the permeability of the vesicular bilayer membrane. At temperatures below Tc, these vesicles were found to be extremely permeable to europium (III) ions. The ion translocation was found to be too fast to be measured by the NMR method under these conditions. However, above the phase transition temperature the ionic permeability decreases to a rate which could be conveniently monitored, and the permeability was shown to increase with temperature and lysolecithin concentration. Analysis of the lysolecithin concentration dependence suggests the formation of ion channels within the lipid bilayer involving four lysolecithin molecules. The data below Tc suggest a phase separation below the phase transition temperature of the host lipid, leading to the formation of patches of lysolecithin molecules within the lecithin matrix. These lysolecithin clusters are presumably long-lived under these conditions and are sufficiently structurally perturbed or disordered to serve as channels for rapid ion permeation.


Oxidative titrations of reduced cytochrome aa3: anisotropic extinction behavior observed in the heme alpha-band region.

Direct chemical titrations of reduced, purified cytochrome aa3 were monitored at 604 nm. Anaerobic oxidation by potassium ferricyanide or 1,1'-bis(hydroxymethyl)-ferricinium ion was compared with the natural substrate, molecular oxygen. The four electrons from reduced cytochrome aa3 were donated to one oxygen molecule, resulting in a linear titration with only fully oxidized or fully reduced enzyme molecules present. Unlike the linear or sigmoidal titrations which resulted from various reductive titrations reported previously, pronounced hyperbolic curves were obtained with the chemical oxidants. The midpoint potential values exhibited by the four metal centers of cytochrome aa3 were determined by computer simulation of the titration curves. A high potential pair of components (heme a = 340 mV, Cu = 340 mV) and a low potential pair (heme a = 220 mV, Cu = 240 mV) were observed, consistent with literature values. Unique to these equilibrium studies was a split in the heme a extinction coefficients at 604 nm. The low potential heme a component contributed 80-85% to the total absorbance change. A polarographic assay was used to verify that the integrity of cytochrome aa3 remained intact during equilibrium titrations spanning several hours. These experimental results indicate that simple chemical reversibility does not exist for cytochrome aa3 under anaerobic conditions.


An endonuclease from calf liver specific for apurinic sites in DNA.

An endonuclease specific for apurinic sites in double-stranded DNA has been partially purified from calf liver extracts. The enzyme has a pH optimum of 9.5, is only slightly stimulated by low concentrations of Mg2+, and has a molecular weight of 28 000. Inhibitors of the endonuclease include Ca2+, EDTA, p-HOHgBzO, NaCl, and tRNA. The enzyme introduces single- and double-stranded breaks in depurinated DNA. High concentrations of the enzyme preparation degrade untreated single-stranded DNA, but not ultraviolet (UV) irradiated DNA or DNA treated with methylmethanesulfonate or 7-bromomethyl-12-methylbenz[a]-anthracene. Enzymatic incisions produce 3'-hydroxyl and 5'-phosphate end groups. Some of the properties of the calf liver apurinic endonuclease differ from those of a similar endonuclease obtained from calf thymus by S. Ljungquist and T. Lindahl [(1974), J. Biol. Chem. 249, 1530] and in this laboratory. The data suggest that these are isozymes.


Resolution of ribonucleic acids by Sepharose 4B column chromatography.

Ribonucleic acids were resolved by molecular sieve chromatography on columns of Sepharose 4B. The elution positions of messenger ribonucleic acids were determined by detection of polyadenosine tracts and by support of protein synthesis in a messenger-dependent cell-free system. The elution position of other ribonucleic acid species from the Sepharose 4B was determined by formamide-sucrose density gradient centrifugation. Resolution of ribonucleic acids by this column was not dependent on molecular weight but rather on other properties such as secondary structure or the presence of poly(adenylic acid). The elution profiles of ribonucleic acids on cross-linked Sepharose 4B differed markely from those on conventional Sepharose and appeared to depend on molecular size alone. There was diminished resolution of high molecular weight ribonucleic acids on such columns.


Identification and isolation of protamine messenger ribonucleoprotein particles from rainbow trout testis.

Treatment of rainbow trout testis polyribosomes with ethylenediaminetetraacetic acid released polyadenylated protamine messenger RNA in the form of a ribonucleoprotein (mRNP) particle. This mRNP particle which sedimented at 12-14 S could be identified by hybridization to [3H]poly(U) and was partially purified by two successive sucrose gradient sedimentations. When RNA was extracted from the mRNP particle and used as a template in the wheat germ cell-free protein synthesizing system the sole product of translation was protamine. Of this RNA, 30% contained poly(A) sequences and was shown to comigrate with polyadenylated protamine messenger RNA during polyacrylamide gel electrophoresis. When the proteins of the mRNP particle were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two prominent polypeptides with apparent molecular weights of 73 000 and 29 000 appeared reproducibly. Treatment of trout testis polyribosomes with puromycin in the presence of 0.5 M KCl released a smaller (8-10S) mRNP particle which was similarly shown to contain protamine messenger RNA. Trout testis postribosomal supernatant fraction possessed 16-18S mRNP particles containing polyadenylated RNA which cosedimented with protamine messenger RNA when the particles were dissociated with sodium dodecyl sulfate.


Crystallographic studies of protein denaturation and renaturation. 2. Sodium dodecyl sulfate induced structural changes in triclinic lysozyme.

Cross-linked triclinic lysozyme was denatured with sodium dodecyl sulfate. Removal of the denaturant resulted in a refolding of the protein to a conformation similar to but not identical with the native one. Three-dimensional x-ray diffraction data out to 3.2-A resolution were collected for two states in the refolding pathway, and appropriately weighted electron density difference maps were constructed. An analysis of these maps reveals that a sodium dodecyl sulfate molecule is trapped in the interior of the protein, and results in a separation of regions of the polypeptide chain. Our results are discussed in terms of current models for protein folding.


Selective covalent binding of methionyl-containing peptides and proteins to water insoluble polymeric reagent and their regeneration.

A polymeric reagent of the type P approximately NHCOCH2Cl (where P is Bio-Gel P-100) was prepared. This polymer covalently bound peptides and proteins specifically at methionine residues under acidic conditions in the presence of a small amount of sodium iodide. Treatment of the polymer-peptide conjugate with 2-mercaptoethanol resulted in essentially complete removal of the peptide with regeneration of intact methionyl residues. In an alternative way, the polymer was suspended for 2 h in boiling water. This treatment resulted in the conversion of the bound methionyl residues to homoserine residues and cleavage and liberation of the bound peptides. The polymeric reagent was successfully applied to the separation of methionyl peptides from peptide mixtures and for specific covalent binding of enzymes and biologically active proteins via their exposed methionyl residues, with the retention of their biological activity.