Ruptured renal artery aneurysm in pregnancy. Case report. A patient who survived rupture of a renal artery aneurysm in pregnancy is described. The aneurysm was caused by fibromuscular dysplasia of the renal artery.


Home and hospital confinement in Newcastle upon Tyne, 1960 to 1969. During the decade 1960 to 1969, perinatal mortality rates in Newcastle upon Tyne fell in parallel with national trends, in association with a marked reduction of domiciliary midwifery. Analysis of the records of women booked for confinement at home or in specialist hospitals showed that the reduction of mortality occurred with unexpected uniformity in both categories, in low risk as well as high risk patients, and in all causes of mortality except congenital malformations. It could not be attributed to improvements in maternal characteristics nor to increased size of babies at birth. The most probably explanation seems to be a combination of many improvements in the quality of care, with increased awareness of risks, better selection of high-risk groups, and improved supervision and management throughout. There is no indication that single factor in obstetric management, such as more intervention during labour, had a dominant effect.


North-east Essex perinatal mortality survey--1971 to 1975. A method of carrying out a Perinatal Mortality Survey in a non-academic district obstetric service is described. The results show a trend of improving perinatal mortality and the various factors that may be influencing this trend are discussed.


Erythrocyte 2,3-diphosphoglycerate and associated haematological parameters during the menstrual cycle and pregnancy. Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and associated haematological parameters were measured throughout the normal menstrual cycle, in each trimester of pregnancy and on the third day post partum. There was no significant change during the menstrual cycle but the 2,3-DPG concentration was increased early in pregnancy, and thereafter gradually decreased. Although other metabolic changes are likely to contribute to the increase in 2,3-DPG concentration, it is concluded that 'functional anaemia', secondary to increased oxygen demand, is a more important stimulus than the decrease in haemoglobin concentration. The implications of a decreased oxygen affinity of haemoglobin, secondary to the rise in 2,3-DPG concentration, are discussed in relation to anaemia and blood transfusion in pregnancy.


Ferritin as an assessment of iron stores in normal pregnancy. In Glasgow, the iron stores in healthy women and in normal pregnant women on prophylactic iron supplementation have been assessed by measurement of plasma ferritin. Ferritin concentration fell progressively to a low level in late pregnancy suggesting that the average iron store was inadequate to meet the demands of pregnancy and that dietary iron supplements were therefore required. There was no evidence that iron supplementation led to excessive iron stores.


Pregnancy heartburn in Nigerians and Caucasians with theories about aetiology based on manometric recordings from the oesophagus and stomach. A study by questionnaire of the incidence of pregnancy heartburn in two groups of Nigerians and one group of Caucasians showed an incidence of 9-8 per cent in all Nigerians as against 78-8 per cent in Caucasians. The difference was highly significant (chi 2 = 102-75; P less than 0-0001). An oesophageal manometric study of the lower esophageal sphincter in 12 non-pregnant women and in 12 pregnant Nigerians (10 without heartburn and 2 with heartburn) showed that the mean lower oesophageal sphincter pressure (+/- SD) in the pregnant patients without heartburn was 20-1 +/- 7-0 mm Hg as against 17-9 +/- 7-0 in the non-pregnant women. While one pregnant subject without heartburn had a lower oesophageal sphincter which was partially displaced into the thorax, none of the non-pregnant women had such sphincter displacement. The two pregnant women with heartburn had low resting sphincter pressures and in both of them the sphincters were partially in the thorax. It is argued that pregnancy heartburn is due to the displacement of the lower oesophageal sphincter into the negative pressure environment of the thorax where the sphincter is less capable of resisting reflux. It is also argued that pregnancy heartburn is commoner in Caucasians that in Nigerians because the spincter in non-pregnant Caucasians is often partly intrathoracic and is thus more easily displaced completely into the thorax by the pregnant uterus.


Large airways function during normal pregnancy. Specific airways conductance and forced expiratory volume in one second were measured monthly from the first trimester until term in 30 subjects in the course of a normal pregnancy. Measurements were also made in the postpartum period. There was no significant change in these measurements either throughout pregnancy or compared with postpartum values. Our results suggested that the function of the larger pulmonary airways was not altered in pregnancy.


A multifactorial survey of neonatal juandice. Concern about a neonatal jaundice rate of 8-6 per cent prompted a retrospective survey of 981 full term infants. There was a highly significant association between increased oxytocin dosage and neonatal jaundice in induced labours. A significant association was also demonstrated between neonatal jaundice and both breast-feeding and minor infections. No association was demonstrated between neonatal jaundice and the method of delivery of birth weight. The results of the survey suggest that while oxytocin in high doses should be used with caution, the benefits obtained from the drug outweigh the risk of hyperbilirubinaemia which it may cause.


Intercellular spaces and cell junctions in endometrial glands: their possible role in menstruation. Intercellular spaces and cell junctions in human endometrial glands were studied by elctron microscopy following different methods of fixation. The size of the spaces, known to alter during the cycle, was found to depend also on the method of fixation. Various intrusive cells and different types of cell debris were seen in these spaces. The three most widely recognized cell junctions (desmosomes, tight junctions and gap junctions), were all most abundant near the cell apices. The possible significance of these findings in elucidating the cellular mechanism of menstruation is discussed.


A simple and sensitive radioimmunoassay for pregnancy-specific beta1-glycoprotein. A simple, sensitive and relatively rapid radioimmunoassay for pregnancy-specific beta1-glycoprotein (PSbetaG) is described. The concentrations of PSbetaG in plasma from cord blood and in amniotic fluid were approximately 0-1% and 1-0% respectively of the maternal plasma levels. The amniotic fluid PSbetaG levels in pregnancies associated with an anencephalic fetus were not significantly different from normal levels.


A review of 50 pregnant patients with kyphoscoliosis. An analysis of the obstetric and medical complications in 50 pregnant kyphoscoliotic patients is presented.


A study of plasma diazepam levels in mother and infant. A 10 mg dose of diazepam was given intravenously to mothers at 15 to 205 minutes before delivery, and plasma diazepam concentrations were measured by gas-liquid chromatography in mothers and infants at delivery and again 24 hours later. The plasma levels in the infants were always significantly higher than in the mothers but there was no evidence to suggest that the newborn were unable to metabolize the drug. All infants had a good Apgar score at birth.


Blood coagulations studies in pregnant patients with infective hepatitis. Coagulation studies were made on 22 pregnant women with acute infective hepatitis and on 15 normal control pregnant women in third trimester. Fourteen hapatitis patients had clinical evidence of liver failure and all of them had a haemorrhagic diathesis; none of the patients without liver failure showed clinical evidence of haemostatic defects. Coagulopathy was present in most patients, its severity being greater in those with hepatic failure. Significant alterations were observed in bleeding time, whole blood clotting time, prothrombin time, thrombin time, plasma fibrinogen and serum levels of fibrinogen degradation products. The last three disturbances were most frequent in patients with liver failure. Increased fibrinolysis and disseminated intravascular coagulation also appeared to play a contributory role, particularly in patients with hepatic failure.


Characteristics of normal labour among different racial groups in Malaysia. The characteristics of normal labour in 977 Malay, Chinese and Indian parturients were established from a retrospective study. Indian babies were found to be significantly smaller than Malay babies which were significantly smaller than Chinese babies (P less than 0-05, P less than 0-05). The mean duration of the first stage of labour taken from the time of admission to the labour ward was 3-4 hours in primiparae and 2-7 hours in multiparae. The mean durations of the second stage of labour were 23-7 minutes and 13-1 minutes respectively. Curves of mean dilatation of cervix and probit analysis at 80% revealed significant differences in the progress of normal labour in primiparae among the three racial groups. The Indian primiparae not only had a slower rate of cervical dilatation but seemed to reach the accelerated phase of dilatation later. No significant differences were noticed in the labours of multiparae.


The relation between umbilical cord tissue prostaglandin E2 levels, mode of onset of labour, fetal distress and method of delivery. The tissue concentrations of prostaglandin E2 (PGE2) were measured by radioimmunoassay in components of the umbilical cord. The levels were significantly higher in the walls of arteries than in non-vascular tissue from the umbilical cord. The highest values were found after emergency Caesarean sections and the lowest after induced ending in normal delivery. The use of forceps and the presence of fetal distress were both associated with significantly raised tissue concentrations of PGE2.


Changes in the concentration of cortisol in amniotic fluid after intra-amniotic prostaglandin for midtrimester abortion. The effects of intra-amniotic prostaglandin E2 (PGE2) and PGF2alpha on the concentration of cortisol in amniotic fluid and in maternal peripheral plasma of patients undergoing abortion between 16 and 23 weeks gestation have been examined. There was a significant (P less than 0-01) increase in the concentration of cortisol in amniotic fluid for hours after PGE2, but not after PGF2alpha. The concentration of cortisol in the maternal peripheral plasma increased in both groups with the progression of abortion, but the levels were not significantly correlated with those in amniotic fluid. These results suggest that by mid-pregnancy fetal cortisol production can be stimulated by PGE2 but the mechanism of this action remains to be elucidated.


Plasma oestrogens in a pregnancy associated with chronic haemodialysis. Pregnancy in a patient undergoing regular haemodialysis at home is described. The pregnancy was complicated by antepartum haemorrhage due to a Type I placenta praevia, and premature labour occurred at 32 weeks, resulting in spontaneous vaginal delivery of a live infant which survived. Plasma progesterone oestrone, unconjugated oestradiol and oestriol levels were normal during the last two weeks of pregnancy, but failed to show a characteristic fall in the puerperium. The conjugated oestriol fraction was 20 to 30 times the normal mean level and did not fall after delivery. These findings are discussed.


The effects of intramuscular norethisterone oenanthate used as a contraceptive on intravenous glucose tolerance and on blood coagulation factors VII and X. Intravenous glucose tolerance tests, and estimations of prothrombin time, and plasma coagulation factors VII and X, were made on 12 women receiving 200 mg norethisterone oenanthate intramuscularly as a contraceptive. These estimations were made before, and at 2 and 12 weeks after, a single injection of norehisterone oenanthate. There was no significant effect on the intravenous glucose tolerance, prothrombin time, or on plasma coagulation factors VII and X, further confirming the safety of norehisterone oenanthate when used as a short-term injectable contraceptive.


Primary echinococcal infection of the ovary. An Iranian Moslem patient with primary echinococcal infection of the ovary is described. Echinococcal infection is extremely rare in practising Moslems.


Primary splenic pregnancy. Case report.

Primary splenic pregnancy. Case report. A patient with a primary splenic pregnancy is described.


Rapid B-scanning of the vitreous.

Rapid B-scanning of the vitreous. Ultrasonic examination is an essential investigation in most patients awaiting vitrectomy. Rapid B-scanning of the vitreous is described utilising a new system capable of high resolution and good tonal quality. Several patterns of haemorrhagic invasion of the vitreous cavity are illustrated, together with detachment, collapse, and retraction of the vitreous gel.


Retinal mobility and retinal detachment surgery. A series of 200 consecutive retinal detachments was examined prospectively to consider the physical sign of mobility of the detached retina. Retinal mobility was found to be absent in 28 cases, and this immobility is caused by periretinal membrane formation. The importance of retinal mobility when considering the case for non-drainage retinal surgery has been examined with particular emphasis on the tear/buckle relationship at the end of the operation. It was found that there was an excellent prognosis (92% success rate) for cases in which the retina was found to be mobile in the vicinity of the retinal tear, and a high proportion of these cases (71%) can be successfully treated with a non-drainage operation.


Fluorescein pupillary flow in aphakics with intact and spontaneous openings of the vitreous face. The passage of fluorescein dye, injected into the general circulation, from the posterior to the anterior chamber was studied in 60 selected aphakic eyes between 2 days and 4 weeks after an uneventful cataract extraction. In a group of 22 eyes with intact anterior hyaloid, fluorescein was seen to pass only through the pupillary margin, mostly within 20 seconds of the beginning of the eye injection. In 24 eyes with rents in the anterior hyaloid the dye was seen percolating only through the vitreous face openings. No fluorescein passed through the pupillary margin or the peripheral iridectomy in this group of eyes. The appearance time of the dye was delayed to about twice the normal appearance time. In 8 eyes with rents in the anterior hyaloid the dye passed only through the pupillary margin. In 4 eyes with anterior vitreous rents the fluorescein appeared through the anterior vitreous face opening. From the analysis of the data it appears that the spontaneous breaks in the anterior vitreous face may represent a 'self-c,re' of pupillary and iridectomy block by the vitreous in aphakics in a high proportion of cases. It is our impression that 3 peripheral iridectomies and a very tight closure of the wound can prevent the complication of spontaneous breaks of the vitreous face.


The asymmetrical visual evoked potential to pattern reversal in one half field and its significance for the analysis of visual field defects. The consistency of the major positive component (P100) of the full-field pattern-reversal response provides a clinically valuable and objective means of detecting visual field defects. Its normally symmetrical distribution about the midline of the occipital scalp results from the summation of two highly asymmetric half-field responses, each of which shows the positive component well lateralised with a widespread distribution on the ipsilateral side. Stimulation of each eye in patients with bitemporal and homonymous hemianopias results in two characteristic patterns of asymmetry, named 'crossed' and 'uncrossed' respectively, in which the major positivity is consistently recorded on the side ipsilateral to the preserved half field. Recordings from a patient after occipital lobectomy confirm the authors' previous suggestion that although the major positive component is recorded from the ipsilateral scalp the typical asymmetric half-field response is generated in the contralateral hemisphere.


Pseudoexfoliative disease of the lens: a study in electron microscopy and histochemistry. The anterior lens capsule in pseudoexfoliative (PE) disease has been investigated with scanning electron microscopy and transmission electron microscopy. It shows several distinct zones which correlate with those seen clinically. True exfoliation of the anterior lens capsule is shown as an integral pathologic feature of PE disease. PE fibrils are mixed with various degraded cell products, including organelles, of which uveal pigment granules are the most frequent. Sheets of degenerating iris pigment epithelium as well as individual cells are often firmly adherent to the pre-equatorial lens capsule and zonules. PE fibrils on the capsule are 35 to 40 nm in width, often showing several subunit filaments about 10 nm wide. PF filaments forming vertical bundles in the pre-equatorial capsule are 10 nm wide, but show little tendency to aggregate laterally. Histochemical and ultrastructural properties of both types of PE fibril and those of the capsular inclusion suggest that they are closely related fibrillar proteins. PE fibrils share some of the characteristics of amyloid but are not identical substances. Nevertheless, a similar mechanism of synthesis is suggested. The participation of non-lenticular sites in the formation of PE fibrils is not precluded by this hypothesis.


Pseudoexfoliation of the lens in Australian Aborigines. The first cases of pseudoexfoliation of the lens to be found in Australian Aborigines are reported. The prevalence rate in the communities in central Australia is 1-3% and for those 61 years and older is 16-3%. Within the limits of our assessment glaucoma was an associated finding in 8-1% of these cases. A high incidence of old trachoma, previous trauma, and Labrador keratopathy was also noted in these people.


The Elidice: new instrument for visual acuity self-screening. A compact electronic device which enables the user to determine for himself whether his visual acuity reaches a chosen standard has been constructed at a cost of 25 pounds. The instrument provides a test which requires the observer to respond to the orientation of a sequence of red Landolt-Cs and which takes under five minutes to complete. 'Crowding' effects associated with conventional letter charts are avoided and the test does not discriminate against astigmatic observers. One hundred observers were tested on both the new instrument (the Elidice) and a standard Snellen chart under clinical conditions. The overall agreement between the two tests was 87.8%, with the  Elidice having an over-referral level of 10-7% and an under-referral level of 1-5% as compared with the letter chart. This makes the Elidice particularly suitable for the regular screening of laser users for reduced visual acuity due to macular damage.


Band keratopathy with bilateral deafness as a presenting sign of hyperparathyroidism. An unusual case of hyperparathyroidism is reported in which calcification of the cornea was the presenting clinical and diagnostic sign. Early recognition of this condition may be of lifesaving importance.


Colchicine suppression of corneal healing after strabismus surgery. Two patients who had previously undergone uneventful operations for strabismus showed healing of corneal dellen and erosion after withdrawal of colchicine therapy. It is suggested that the exhibition of colchicine therapy for familial Mediterranean fever in these two cases was responsible for initial persistence of these two postoperative complications.


Use of the constant infusion technique for measuring rates of protein synthesis in the New Zealand White rabbit. 1. To study the potential of the constant-infusion technique for measuring rates of protein synthesis in New Zealand White rabbits, animals were infused for up to 6 h with radioactively-labelled tyrosine. 2. Labelled tyrosine from plasma and tissues was isolate from labelled metabolites by ion-exchange chromatography. 3. Analysis of serial blood and muscle biopsy samples removed under anaesthesia showed that the specific radioactivity (SR) of the free tyrosine pools reached an approximately constant value within 2 h. 4. Certain commercial preparations of L-[side-chain 2,3-3H]tyrosine were contaminated with 300 mg radioactive D-tyrosine/g. The D-isomer appeared to enter the muscle intracellular pool. 5. In constant-infusion experiments L-[3H]tyrosine could replace the uniformly-14C-labelled L-isomer for the determination of rates of protein synthesis in muscle. L-[side-chain 2,3-3H]tyrosine may not be suitable for use as a precursor for measuring rates of liver protein synthesis. 6. Evidence is presented that the precursor of liver protein synthesis may not be well defined by the SR for free tyrosine of the homogenate. 7. The technique was used to measure the rates of protein synthesis in adult rabbits. The rates of protein synthesis in liver and muscle were measured and from measurements of tyrosine flux the mean rate of whole-body protein synthesis was calculated as 13-8 g/kg per d.


Determination of rumen microbial growth in vitro from 32P-labelled phosphate incorporation. 1. The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with 32P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth. 2. Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80% of the values obtained using the isotope method for rumen fluid and WCS respectively. It is suggested that results obtained using the former method reflect 'net growth' of micro-organisms which is the result of simultaneous growth and degradation. The isotope method measures 'total growth', as isotope incorporation is not affected by degradation of non-growing cells. 3. Incorporation of 32P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. The results showed different specific rates of synthesis and degradation for all components studied. It is concluded that the composition of microbial matter changed during growth. 4. When N incorporation, calculated from results obtained using the isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18-3 and 44-6 g N incorporated/kg of organic matter fermented were obtained. Low values were associated with large proportions of the substrate being fermented to lactate and the use of glucose instead of disaccharides as substrate. Part of the variation could also be attributed to differences in incubation period, reflected in different proportions of polysaccharide formed. 5. The use of isotopes for determination of rumen microbial growth in vitro is critically discussed.


The influence of alpha-linolenic acid (18: 3omega3) on the metabolism of gamma-linolenic acid (18: 3omega6) in the rat. 1. Essential fatty acid-deficient rats were fed gamma-linolenic acid (18: 3omega6) at 2% dietary energy and alpha-linolenic acid (18: 3omega3) at 0, 1-6, 2-8 and 4-0% of the dietary energy. 2. 18: 3omega3 at 1-6% apparently inhibits the synthesis of the C20 and C22 omega6 long-chain polyunsaturated fatty acids (omega6 LC-PUFA) metabolized from 18: 3omega6. 3. However, increasing the dietary levels of 18: 3omega3 from 1-6 to 4-0% has no further influence. 4. The results suggest that dietary 18: 3omega6 is an efficent precursor for the omega6 LC-PUFA synthesis even in the presence of 18: 3omega3.


Dry matter and nitrogen in the duodenal contents of growing pigs: a discrepancy explained. 1. Discrepancies between the results obtained from experiments on pigs with re-entrant duodenal cannulas have been investigated with reference to diet, cannula site and collection procedure. 2. The results obtained from a sealed automatic collection system were different from those obtained from an open manual method and it was concluded that the maintenance of gut intra-luminal pressure in the automatic system was the causative factor.


Fractional flux rates of Nt-methylhistidine in skin and gastrointestine: the contribution of these tissues to urinary excretion of Nt-methylhistidine in the rat. 1. Fractional flux rates of Ntau-methylhistidine (3-methylhistidine; Me-His) of skin and gastrointestine were measured by administering [methyl-3H]methionine to rats. 2. The results showed that the contribution of these tissues to urinary excretion of Me-His was at least 16-6%. This means that when fractional catabolic rates of myosin and actin were estimated from urinary excretion of Me-His, the part of Me-His derived from skin and gastrointestine should not be neglected.


The susceptibility of suckling lambs to zinc toxicity. 1. Suckling lambs maintained for 4 weeks on a milk-substitute diet containing 407 g Toprina yeast/kg dry matter of diet exhibited poor growth, low appetite and extensive renal damage. 2. The batch of yeast used contained 2065 mg zinc/kg. 3. The kidneys of lambs offered a milk diet supplemented with the same Zn content as the yeast-fed diet (32-1 mg Zn/1000 kJ) were similarly affected.


Metabolic studies in rats of 75Se incorporated in vivo into fish muscle. 1. [75Se]selenite of [75Se]selenomethionine was injected in to the coelomic cavity of fish. After 2 d or 14 d the muscle portion of the fish was removed and homogenized. The long-term fate in rats of an oral dose of each labelled homogenate was compared with that of an oral dose of [75Ee]selenite or [75Se]selenomethionine mixed with unlabelled fish homogenate. 2. Urinary and faecal radioactivity were measured during the 1st week and whole-body radioactivity was determined for 10 weeks. Rats were killed at weekly intervals for 4 weeks for analysis of tissue distribution of 75Se. 3. Intestinal absorption of 75Se given as labelled fish homogenate was less complete than that of 75Se mixed with unlabelled homogenate, and the absorption of 75Se from the 14 d-labelled fish homogenate derived from [75Se]selenite was less complete than that of 75Se from the other labelled homogenates. 4. Urinary excretion of absorbed 75Se in the first 7 d was in the range 5--8% absorbed dose and was slightly greater in the rats given 75Se as selenite or derived from selenite than in those given 75Se as selenomethionine or derived from selenomethionine. Endogenous faecal excretion of absorbed Se was similar in all groups, as also were tissue distribution of retained 75Se and long-term whole-body turnover rate. 5. The results of these studies are compared with those of earlier studies of the metabolism in rats of [75Se]selenomethionine, [75Se]selenite, [75Se]selenocystine and 75Se incorporated in vivo into rabbit kidney. There were differences in the initial utilization of 75Se from these various sources but after the 1st week 75Se from all sources appeared to be metabolized similarly, suggesting that for rats dietary Se of all forms is ultimately incorporated into the same metabolic pool.


Daily intakes of manganese, copper, zinc and cadmium by New Zealand women. 1. A total of 179 duplicate diets were collected by twenty-three women consuming their habitual diet. Twelve of the subjects were living in a residential hall for students, the others were living in their own homes or in flats. Collection periods varied from 3 to 21 d. 2. Average daily intakes were: manganese 2-7 mg; copper 1-5 mg for diets not containing liver, 7-6 mg for fifteen diets containing liver; zinc 10-0 mg; cadmium 21 microgram for diets not containing liver, 27 microgram for fifteen diets containing liver. 3. The intakes were compared with values for adults from other countries and with current recommended dietary allowances or estimated minimum requirements. Mn intakes were typical for non-vegeratian Western diets. Cu and Zn intakes may have been marginally low. The intakes of Cd were low. 4. Daily intakes of protein (71 g), fat (83 g), fat (83 g), carbohydrate (224 g) and energy (8-1 MJ) from 129 of the diets were similar to recent values obtained for New Zealand women. 5. Subjects living in the residential hall had higher intakes of all nutrients than subjects living in their own homes or flats.


The effects of various forms of gastrointestinal cannulation on digestive measurements in sheep. 1. There was little difference in digestive (voluntary food intake, dry matter digestibility and nitrogen balance) and blood measurements (venous concentrations of corticosteroids, serum aspartate aminotransferase (EC 2.6.1.1), protein-bound iodine, urea and glucose) of intact sheep (eight animals) and of sheep prepared with rumen cannular (sixteen animals) and subsequently with either simple 'T-shaped' (eight animals) or re-entrant cannulas (eight animals) at the duodenum and ileum, when fed ad lib. a chopped medium-quality-hay ration. 2. Wool growth rates of the intact sheep were similar to those in sheep with rumen cannulas and with rumen cannulas plus simple 'T-shaped' cannulas, but higher (P less than 0-01) than those with rumen cannulas plus re-entrant cannulas. 3. When the sheep were subsequently given a restricted intake (800 g/d) of dried grass, retention times of solid- and liquid-phase digesta markers in the rumen and caecum were similar in all sheep. 4. The use of the different preparations in digestive physiology studies is discussed.


Polyamines and their biosynthetic decarboxylases in various tissues of the young rat during undernutrition. 1. Male weanling rats were maintained at a constant body-weight by feeding them reduced amounts of the normal diet for various periods up to 4 weeks. Control male rats were allowed free access to the normal diet and some were killed at the beginning of the experiment and others at the same ages as the experimental rats. 2. After killing by cervical dislocation the rats had their liver, quadriceps muscles and spleen removed. The tissues were weighed and the activities of the enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMD; EC 4.1.1.50) assayed in each tissue. In the liver the content of the polyamines (spermidine and spermine) and putrescine was also measured. 3. The liver and quadriceps muscles showed an over-all maintenance of weight during undernutrition, but the spleen lost weight during the first 7 d of undernutrition and then remained constant. The weight of the liver increased by approximately 50% following the daily maintenance feed, but returned to its prefeeding value by 24 h after feeding. 4. During the first 7 d of undernutrition ODC activity decreased in all three tissues, and remained fairly constant thereafter. In the liver there were marked increases in the activity of ODC during the first 4 h after the daily feed, but the activity then decreased to prefeeding values. SAMD activity tended to remain normal in the liver, decreased initially and then returned to normal in the quadriceps muscles, and remained normal initially and then decreased in the spleen. Hepatic SAMD activity showed no consistent response to the daily feed, but quadriceps SAMD activity increased significantly between 1 and 8 h after feeding. 5. Hepatic putrescine content remained constant during undernutrition whilst spermine increased slightly and was then maintained above normal for liver size. Hepatic spermidine content decreased initially and then remained constant. Putrescine increased slightly in response to the daily feed and spermidine increased considerably. Spermine content was unaffected by the daily feed. 6. It is suggested that the response of polyamine synthesis in the various tissues is primarily dependent upon the way in which nutrients are made available to the tissues. The maintenance of spermine content in the liver at the expense of spermidine may be related to differential changes in the nucleic acids.


Distribution of copper and zinc in the liver of the developing sheep foetus. 1. A study has been made of the copper- and zinc-binding proteins in the livers from sheep foetuses of 80-142 d gestational age. 2. Metallothionein was found to constitute the major Zn-binding component in the cytosol at all times and to be identical to Zn-thionein from adult sheep liver. 3. Zn also occurred in two fractions, not normally found in sheep liver, with approximate molecular weights of 28000 and 47000. The relative proportions of these were age-dependent. 4. Between 15 and 35% of the hepatic Cu, corresponding to most of the Cu in the cytosol, also occurred in the metallothionein-containing fraction.


The effect of dietary copper sulphate on laying performance, nutrient intake and tissue copper and iron levels of the mature, laying, domestic fowl. 1. A cereal-based diet containing 16 mg copper/kg was fed ad lib. to a group of laying hens for 35 d. Five other groups were given this control diet to which was added 120, 240, 480, and 1920 mg Cu/kg (as copper sulphate). 2. Records were kept of daily food intake, water intake and egg production. 3. After 35 d the hens were slaughtered and blood haemoglobin, packed cell volume, Cu and aspartate aminotransferase (EC 2.6.1.1) levels assayed. Liver, oviduct, kidney and breast muscle Cu and iron concentrations were measured. 4. Food and water intakes were depressed by the two highest levels of dietary Cu and water intake was increased by the diet with 240 mg added Cu/kg. Both food and water intake showed a quadratic relationship with the level of added dietary Cu. 5. Body-weight loss was increased by the addition of Cu and showed a significant linear relationship with the concentration of added Cu in the diet. Liver and oviduct weights were depressed at the two highest levels of Cu addition. 6. Liver and oviduct Cu and Fe concentrations were significantly increased by high dietary Cu and mean total liver and kidney Cu and Fe showed an increase although for the liver this was not statistically significant.


Photoaffinity labeling of acyl-coenzyme A:glycine N-acyltransferase with p-azidobenzoyl-coenzyme A. A photolabile reagent, p-azidobenzoyl-CoA, has been synthesized and tested as a photoaffinity label for acyl-CoA:glycine N-acyltransferase (EC 2.3.1.13) from beef liver. p-Azidobenzoyl-CoA is an active-site-directed reagent for this N-acyltransferase, since it is an alternate substrate (Km = 26 micronM, when [glycine] = 100 mM). Ultraviolet irradiation of a mixture of p-azidobenzoyl-CoA and the N-acyltransferase produces irreversible inhibition. Benzoyl-CoA protects the enzyme from inhibition by photoactivated p-azidobenzoyl-CoA. Acyl-CoA:glycine N-acyltransferase is composed of a single polypeptide with a molecular weight of about 35 000. Photolabeling experiments show that there is one active site per molecule of enzyme.


A nuclear magnetic resonance study of sphingomyelin in bilayer systems. The physical properties of small single-walled vesicles composed of the zwitterionic phospholipid sphingomyelin have been studied using 1H and 31P nuclear magnetic resonance spectroscopy. The temperature variation of proton line widths and spin-lattice relaxation times and the chemical shift behavior for sphingomyelin vesicles are compared with results previously determined for phosphatidylcholine vesicles. Differences between the two systems are interpreted as indications of the presence of both inter- and intramolecular hydrogen bonding in sphingomyelin bilayers.


More on the motional state of lipid bilayer membranes: interpretation of order parameters obtained from nuclear magnetic resonance experiments. Proton and deuterium order parameters measured for the liquid crystalline phase of unsonicated lipid bilayer membranes are interpreted in terms of two motions: (i) chain reorientation and (ii) chain isomerization via kink diffusion. The observed order parameters are found to be compatible with angular deflections of the chain of about 50 degrees with respect to the bilayer normal, coupled with a probability of trans orientation of a methylene segment in the upper part of the chain of about 0.8-0.9. The motional model can be shown to account for the dynamic properties of the membrane system as measured by nuclear magnetic relaxation measurements, assuming that the chain isomerization occurs at a rate of approximately 10(10) s-1 and chain reorientation at a rate of approximately 10(7) s-1. Analysis of proton and deuterium line-width data in terms of this model shows that sonication has the effect of increasing the rate and amplitude of chain reorientation without substantially changing the isomerization motion along the acyl chain. These conclusions are briefly compared with similar observations recently reported in Raman spectroscopic studies.


A simple method for the preparation of homogeneous phospholipid vesicles. A new method is described for the preparation of homogeneously sized, single-lamellar phospholipid vesicles. This method, wich is based on differential high-speed ultracentrifugation, has the advantages of a higher vesicle yield without dilution and rapidity of preparation when compared to the molecular-sieve technique. The homogeneity of vesicle dispersions, prepared by this new method, is examined by several physical techniques and found to be comparable to the best samples prepared by molecular-sieve chromatography.


Investigation of the structure of bovine erythrocyte superoxide dismutase by 1H nuclear magnetic resonance spectroscopy. The 270-MHz 1H nuclear magnetic resonance spectra of the apo, copper(I)-zinc(II), and copper(II)-zinc(II) forms of bovine erythrocyte superoxide dismutase (EC 1.15.1.1) are reported, and assignments of resonances to ten amino acid residues are proposed. The data require that at least four and probably six histidine residues serve as ligands to the metals in each subunit of the enzyme, consistent with x-ray diffraction results. The remaining assigned resonances are associated with His-19, His-41, Tyr-108, and the N-terminal N-acetyl group. The imidazole C(2)H of His-41 exchanges readily at pH greater than 8. The structural implications of the effect of the paramagnetic Cu(II) in the holoenzyme on the proton relaxation times are in reasonable accord with the data from x-ray diffraction studies.


Template activity of calf thymus DNA modified by a dihydrodiol epoxide derivative of benzo[a]pyrene. The purpose of the present study was to determine the effects of covalent binding to DNA of a reactive derivative of benzo[a]pyrene on template activity during in vitro transcription with RNA polymerase. Calf thymus deoxyribonucleic acid, modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, was transcribed with Escherichia coli DNA-dependent RNA polymerase. With increasing levels of modification, there was a progressive inhibition of transcription. The inhibition was much greater under conditions where continuous reinitiation of transcription occurred than under conditions where only one RNA chain was synthesized per initiation site. This suggested that the modified sites block the movement of polymerase along the template and prevent recycling of the enzyme. Consistent with this interpretation were analyses of RNA transcripts on sucrose density gradients which showed a progressive decrease in average RNA chain length as the extent of template modification increased. In contrast to the inhibitory effect on chain elongation, evidence was obtained that the modified DNA had an increase in the number of initiation sites for transcription. These results are consistent with separate physical studies indicating that modification of DNA by this benzo[a]pyrene derivative can induce small localized regions of denaturation.


Changes in polypeptide initiation and elongation rates during the cell cycle of Chlamydomonas reinhardi. Changes in the rate of protein synthesis during the cell cycle of Chlamydomonas reinhardi have been measured by determining changes in the separate rates of polypeptide chain initiation and elongation and in the rate of incorporation of a radioactive amino acid. The rate of polypeptide chain elongation, determined from the relative rates of labeling of two size classes of polyribosomes, varies only about twofold during the cell cycle. The rate of polypeptide chain initiation, determined from an analysis of the distribution of ribosomes in monoribosomes (and ribosomal subunits) and polyribosomes, varies more than 25-fold. Also, the overall rate of protein synthesis during the cell cycle varies to the same extent as the rate of chain initiation. Measurement of protein synthetic rates using incorporation of a radioactive amino acid ([3H]arginine) underestimates the actual change in the rate of protein synthesis during the cell cycle. The vast changes in the initiation rate during the cell cycle suggest a mechanism for selecting specific messenger RNAs for translation at different cell-cycle stages.


Study of DNA synthesis in chromatin isolated from HeLa cells. When incubated in vitro, HeLa cell chromatin can synthesize DNA at rate comparable to that observed with isolated nuclei. The in vitro DNA synthetic activity of chromatin reflects DNA synthesis in intact cells since chromatin from cells in S phase are several times more active thatn preparations derived from mitotic cells. The requirements for the synthesis of DNA by chromatin preparations are also similar to those of isolated nuclei and the size of the DNA pieces made in both systems is roughly comparable. The chromatin system offers several advantages not available with isolated nuclei. Chromatin will synthesize DNA for a much longer time than isolated nuclei so that larger amounts of DNA can be synthesized in vitro In addition, although chromatin has its own endogenous ability to synthesize DNA, it is markedly stimulated by the presence of exogenously added HeLa cell DNA polymerase alpha, beta, and gamma, and, thus, may provide a new template system for the study of DNA synthesis.


Light-activated amino acid transport systems in Halobacterium halobium envelope vesicles: role of chemical and electrical gradients. The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (Vmax) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms; i.e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na+ and K+ are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in  procaryotes.


Subunit structure of the orotate phosphoribosyltransferase--orotidylate decarboxylase complex from human erythrocytes. A complex of orotate phosphoribosyltransferase and orotidylate decarboxylase has been shown to exist in three molecular weight forms (Brown, G. K., Fox, R. M., and O'Sullivan, W. J. (1975), J. Biol. Chem. 250, 7352). The smallest of these, of molecular weight 62 000, was subjected to further study. On the basis of the inactivation of the enzyme activities, carried out in the presence of low concentrations of guanidine hydrochloride, and of changes in molecular weight of preparations during aging, it was inferred that the enzyme complex contained more than one type of subunit. This was confirmed by chromatography on Sephadex G-75 after preincubation in guanidine hydrochloride or with guanidine hydrochloride in the elution buffer. It was concluded that the enzyme complex consisted of two types of subunits, two decarboxylase units of molecular weight approximately 20 000 and two further subunits of approximately 13 000. The subunits could be separated and reassociated with partial recovery of both activities. A 40 000 molecular weight form had full decarboxylase activity but no phosphoribosyltransferase activity. Restoration of the 62 000 molecular weight form resulted in restoration of both enzymatic activities. An intermediate species of molecular weight 50 000 representing a combination of the decarboxylase dimer with one of the 13 000 subunits was also demonstrated. This form required the presence of dithiothreitol in order to manifest phosphoribosyltransferase activity. A model of the system has been proposed that accounts for both the different molecular weight forms and also for the deficiency of both activities in the rare inborn error of metabolism, hereditary orotic aciduria.


Infrared linear dichroism investigations of deoxyribonucleic complexes with histones H2B and H3. Complexes between DNA and histones H2B and H3 were studies by means of infrared linear dichroism in a wide range of histone to DNA ratios and of different relative humidities. The measurement of the dichroic ratios allows one to determine the secondary structure of DNA in the complexes. It is shown that the progressive addition of histone H2B or H3 to DNA inhibits the structural B leads to A transition and DNA remains in a B-type form at low relative humidity. A new simple method is proposed to evaluate the amount of A or B forms of DNA when both structures are present. It is found that the B leads to A transition is fully inhibited when only one molecule of H2B or H3 histone is bound per about three or four turns of DNA helix, respectively. It is proposed that about four to three turns of DNA helix represent the "critical length of DNA" (minimum "cooperative unit") for the B leads to A transition.


Bovine enterokinase. Purification, specificity, and some molecular properties. Enterokinase has been isolated from the contents of bovine duodena and purified 1200-fold in 41% yield. The isolation procedure employed DEAE chromatography, affinity chromatography on immobilized p-aminobenzamidine, and gel filtration. The resultant pure enzyme exhibits a single band on sodium dodecyl sulfate gel electrophoresis corresponding to a molecular weight of 145 000. Two chains in the molecule, connected by disulfide linkages, have molecular weights of 57 000 and 82 000, respectively. The purified enzyme exhibits a restricted specificity which is directed toward the polyanionic amino acid sequence in the activation peptide of the zymogen substrate. Of the zymogens of the serine proteases tested, including several of those involved in blood coagulation, only native and guanidinated trypsinogen are acitvated by enterokinase, whereas acetylated trypsinogen is not. Partial heat denaturation of bovine enterokinase causes a differential response toward the activation of trypsinogen and the hydrolysis of benzoyl-L-arginine ethyl ester (BZArgOEt), further suggesting that secondary sites are important in the binding of trypsinogen. A sensitive assay for enterokinase (nanomole range) was developed using tritiated BZArgOEt.


Calcium binding to rat heart plasma membranes: isolation and purification of a lipoprotein component with a high calcium binding capacity. A calcium binding lipoprotein component of rat heart plasma membrane was isolated, purified, and characterized. The lipoprotein complex had an apparent molecular weight of 71 400. One mole of lipoprotein contained 90 mol of phospholipid and 1 mol of a 12 300 molecular weight protein. The maximum calcium binding capacity was 4.27 mu mol/mg of protein, which corresponded to 52 mol of calcium per mol of lipoprotein complex. Calcium binding was competitively inhibited by a variety of metal ions and experimental antiarrhythmic and anesthetic agents.


Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.


Nanosecond fluorescence spectroscopy of pyrenebutyrate anti-pyrene antibody complexes. The utility of the long-lived fluorophore, pyrene, as a probe in nanosecond fluorescence depolarization measurements was investigated using pyrenebutyrate bound in the combining sites of rabbit antipyrenebutyrate immunoglobulin G. The time dependent anisotropy decay data points showed very little scatter in the time interval 0-350 ns, which is more than three times the comparable time interval observed with epsilon-1-dimethylamino-5-naphthalenesulfonyllysine (DNS-lysine) bound in the combining sites of anti-DNS antibodies [Holowka, D.A., and Cathou, R.E. (1976), Biochemistry 15, 3379]. Thus, the use of pyrene can significantly extend the range of macromolecular rotational correlation times that can be measured by the single photon technique. In the present investigation, we confirmed the presence of Fab segmental fexibility in immunoglobulin G molecules specific for a hapten different from DNS-lysine. We obtained a value of about 135 ns for the longer rotational correlation time which probably represents global rotation of the entire molecule. In the course of these experiments, we have also found that the combining sites of antipyrenebutyrate antibodies are, as expected, relatively nonpolar.


Effect of an anti-tumor platinum complex, Pt(II) diaminotoluene, on mitochondrial membrane properties. The effects of platinum complexes, selected for their potent anti-tumor activities, have been studied on rat liver mitochondria. Among the mitochondrial properties which have been studied, the most marked effects of platinum complexes were obtained on functions linked to the inner membrane. cis-Pt(II)(3,4-diaminotoluene) dichloride is shown to stimulate state 4 respiration. It inhibits the phosphate transport into mitochondria, decreases the accumulation of Ca2+, and induces a more rapid release of the accumulated Ca2+. A release of Mg2+ from mitochondria incubated in the absence of added divalent cations, and an efflux of divalent cations from mitochondrial membranes are also observed. All these results indicate a profound modification of the of the permeability of mitochondrial membrane.


The rise in chlorophyll a fluorescence yield and decay in delayed light emission in tris-washed chloroplasts in the 6-100 microseconds time range after an excitation flash. Parallel measurements of the rise in chlorophyll a fluorescence yield and delayed light emission decay, after a 10 ns saturating excitation flash, have been made in tris (hydroxymethyl)aminomethane-washed chloroplasts. Various electron donor systems (Mn2+; ascorbate; reduced phenylenediamine and benzidine) were used in conjuction with different preillumination regimes to alter [P+-680], the oxidized form of the Photosystem II reaction center chlorophyll a. Conditions giving rise to high [p+ -680] resulted in only a small rise in fluorescence yield, an inhibition of a 6 microseconds component of delayed light emission. These results confirm the hypothesis that P+-680 acts as a quencher of fluorescence and that delayed light emission in the microsecond time range is due to the back reaction of P+-680 and Q-. (Q is the first "stable" electron acceptor of Photosystem II.) Two preillumination flashes are required before the full effect of Tris washing is observed in the delayed light emission decay and fluorescence yield rise; this suggests that a capacity to hold two charges exists between the Tris block and P+-680. Tris washing has no direct effect on the movement of electrons from Z (the first electron donor to P+-680. Finally, Mn2+ donates electrons to P+-680 via Z.


Oligomycin sensitivity of mitochondrial sulfhydryl groups. Dithionitrobenzoate has been used to titrate sulfhydryl groups of rat liver mitochondria in glutamate buffer, pH 7.4. Reaction with oligomycin and different SH reagents preceded the SH titration. Under these conditions it was found that 2-mercaptopropionylcne and N-ethylmaleimide reacted in an oligomycin-sensitive manner, so that the control values (in the absence of SH reagent) were obtained. Similar concentrations of mersalyl and of N-(N-acetyl-4-sulfamoylphenyl) maleimide, in the presence of oligomycin, enhanced reactivity toward Nbs2. The concentration range of oligomycin-sensitive SH groups was thus defined between approx. 5 and 9 nmol reagent/mg mitochondrial protein. In this way, a differentiation between SH grops, which are implicated in phosphate transport antd those, which react in an oligomycin-sensitive manner, and which are probably connected with the coupling mechanism was achieved.


Picosecond and microsecond pulse laser studies of exciton quenching and exciton distribution in spinach chloroplasts at low temperatures. Studies of the fluorescence quantum yield and decay times, determined at the emission maxima of 685 and 735 nm, using picosecond laser pulses for excitation, indicate that the pigments which are responsible for the 735 nm emission derive their energy by transfer of singlet excitons from the light-harvesting pigments and not by direct absorption of photons. Microsecond pulse laser studies of the fluorescence quantum yields at these two fluorescence wavelengths indicate that long lived quenchers (most probably triplet states), which quench singlet excitions, accumulate preferentially within the long wavelength pigment system which gives rise to the 735 nm emission band.


Dipole moment changes and voltage dependent membrane capacity of squid axon. Changes in the membrane capacity of squid axons during hyper- and depolarizations are measured between --160 and +40 mV. After corrections for the series resistance and fringe effect, we found that the membrane capacity increased from 0.68 to 1.2 muF/cm2 with depolarization. It was further observed that tetrodotoxin the external medium eliminated the change in membrane capacity without affecting the conductivity. The voltage-dependent membrane conductivity is, in turn, greatley reduced by the internal cesium ion. These observations clearly indicate that the voltage-dependent membrane capacity and conductivity are closely related to ionic channels. Particularly, the increase in membrane capacity with depolarizations may be due to sodium channels. The change in the dipole moment associated with sodium sites was determined using values of alpham and betam at various depolarizations. We found, based on voltage clamp measurements, that the increase in the dipole moment of the sodium site between --40 and --5 mV is 1230 Debye units (D.U.) and 930 D.U. between --5 and +60mV, indicating that the depolarization of sodium channels may consist of two different steps.


Extensive exchange of rat liver microsomal phospholipids. Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.


Alterations of RNA metabolism in sea urchin embryos by an inhibitor of protein synthesis initiation. An inhibitor of the initiation of protein synthesis, 2-(4-methyl-2,6 dinitroanilino)-N-methyl propionamide, (MDMP), inhibits incorporation of amino acids by 85--95% without altering the rate of incorporation of uridine into sea-urchin-hatched blastula embryos. Since the inhibition is readily reversible up to at least 90 min after addition, extensive secondary effects are unlikely. Newly synthesized RNA accumulates in polyribosome-like structures in amount and with a size distribution very similar to the control. The polyadenylation of total, cytoplasmic and polyribosomal RNA are only slightly different from the controls. While the size distribution of 9-S of approx. 30-S RNA is about the same, there is relatively less low molecular weight RNA (approx. 4-S). There is also a greater accumulation of greater than 30-S RNA in the cytoplasm of treated embryos due either to leakage and/or improper processing of nuclear RNA. The latter possibility is consistent with the decrease in 4-S RNA which is a presumed degradation product of nuclear HnRNA. While the initial rate of RNA synthesis and much of the accumulation of RNA in polyribosomes are not affected, the inhibition of protein synthesis per se or a secondary effect of MDMP results in altered patterns of RNA transport and processing.


Messenger RNA binding of a ribosome-associated protein kinase and a ribosomal phosphoprotein(s) in mouse plasmacytoma. A specific ribosome-associated protein kinase (protein kinase II) and phosphoprotein(s) from the ribosomal KCl wash fraction termed "PPx" have been isolated from plasmacytoma, and tested for their ability to bind to poly(A) and to different plasmacytoma polynucleotides. The nitrocellulose filter binding technique was used to measure RNA-protein interaction. Protein kinase II and PPx preferentially bound mRNA compared to poly(A). They did not bind ribosomal RNA, soluble RNA or DNA. The optimal conditions (temperature, time, protein concentration, ionic strength) for mRNA-protein interaction were determined. Ribosomal protein kinase (protein kinase II) phosphorylated PPx proteins which bound to mRNA represented at least two bands as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Mr = 90 000 and 80 000). The high  affinity of protein kinase II and PPx for mRNA suggests that they may function in regulating protein synthesis.


Flow dichroism, flow polarized fluorescence and viscosity of the DNA acridine complexes. The strong binding of various acridine dyes to DNA has been studied by the measurements of flow dichroism, flow polarized fluorescence and viscosity. Negative flow dichroism and percentage change in polarized fluorescence intensity show that intercalated dye molecules are oriented rather perpendicularly to the main axis of the DNA helix, like base pairs. On the other hand, viscosity measurements show that the increase of the contour length of DNA depends on the dye structure, being much smaller in the case of dyes with bulky substituents compared to that of the other dyes. This may be attributed to the formation of the outside bound complex. Further, the introduction of bulk substituents to the acridine ring leads to a little smaller values of the reduced dichroism and intensity change of polarized fluorescence. The results may be qualitatively understood if we assume that the outside bound dye lies in the groove of the DNA helix and the plane of the dye tilts from the perpendicular direction relative to the main axis of the helix.


High molecular weight DNA intermediates synthesized by permeabilized L cells. Mouse L cells rendered permeable to deoxynucleoside triphosphates synthesize DNA as an extension of replication forks that were active in the intact cells. The permeabilization process does not affect the size of the bulk cell DNA. Intermediate molecular weight DNA, synthesized in the intact cells, is neither degraded to small molecular weight DNA nor processed into bulk cell DNA following the permeabilization process. DNA synthesized by the permeable cells demonstrates a heterogenous distribution in alkaline sucrose gradients, with peaks at 26 S and 71 S. Pulse-chase experiments demonstrate that these two classes of DNA intermediates do not have a precursor product relation. They appear to be synthesized independently and at the same rate. The results are compatible with continuous synthesis of DNA along one template strand and discontinuous synthesis along the opposite strand. DNA synthesis in isolated L cell nuclei was compared to the process in permeabilized cells with the results demonstrating that the rate of DNA synthesis is slower in the nuclei than it is in the permeable cells. Alkaline sucrose gradient studies demonstrate that the DNA synthesized by isolated nuclei is smaller than the DNA synthesized by the permeable cells.


Differential partitioning of tRNAs between micellar and aqueous phases: a convenient gel filtration method for separation of tRNAs. Partition coefficients (Kp) of phenylalanine, glutamic acid-II and tyrosine tRNAs between water and micellar cetyltrimethylammonium chloride (CTACl) have been determined to be 5600, 520 and 3100, respectively, by gel filtration on Sephadex G-100-120. Equations for obtaining Kp values from quantitative gel filtration have also been derived. The use of this method for the separation of tRNAs is demonstrated.


Streptomycin: separation of polysomal and non-polysomal messenger ribonucleoproteins. In the presence of 0.02 M streptomycin, all of the polysomes precipitate from male cricket (Acheta domesticus) accessory gland and chick embryonic tissue post-mitochondrial fractions. All non-polysomal messenger-like ribonucleoprotein preparations tested remain in solution.


Kinked DNA. Energetics and conditions favoring its formation. Theoretical calculations demonstrate that the formation of kinks in DNA in the manner proposed by Crick and Klug produces a conformation which corresponds to a local energy minimum when the phosphate backbone is charged. This conformation becomes the global minimum under certain conditions which neutralize the anionic backbone oxygens. A sequence preference for kink formation is predicted. The possible role of kinking in experimentally observed phenomena and experimental means of demonstrating the existence of kinks are discussed.


Purification and properties of cytochrome p-450 (11beta- and 18-hydroxylase) from bovine adrenocortical mitochondria. We describe an improved procedure for the preparation of a cytochrome P-450 from bovine adrenocortical mitochondria which catalyzes 11beta- and 18-hydroxylation of steroids. The preparation is based upon chromatography on DEAE cellulose which separates the enzyme from the side-chain cleavage P-450, which can also be prepared in highly purified form from the same tissue extracts. The enzyme behaves as a single compound in glycerol density gradients. The enzyme aggregates at protein concentrations greater than 1 mg/ml to a series of forms of various molecular weights. On Sepharose 4B the enzyme shows a molecular weight of 185 000, while on glycerol density gradients a molecular weight of 1 - 10(6) is observed. The subunit molecular weight determined by electrophoresis on polyacrylamide gels with sodium dodecyl sulfate is 47 500 and the protein appears as a single band. The ratio of 11beta-/18-hydroxylase activities does not change significantly during purification and is constant through the protein peak on glycerol density gradients. Since there appears to be only one subunit species, it seems likely that the two hydroxylase activities are catalyzed by one protein.


Inhibition of potato starch phosphorylase by alpha-D-glucopyranose-1,2-cyclic phosphate. alpha-D-Glucopyranose-1.2-cyclic phosphate is a potent inhibitor of potato starch phosphorylase-catalyzed (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) starch elongation. The inhibition is competitive with respect to alpha-D-glucopyranose 1-phosphate (Glc-1-P) with Ki approximately 0.07 mM at pH 6.3 and 30 degrees C in 25 mM citrate buffer. The affinity of the phosphorylase - starch complex for the cyclic ester is therefore nearly 30 times as large as for Glc-1-P. Under conditions where alpha-D-glucopyranose-1,2-cyclic phosphate slows starch elongation by a factor of 3, UDPglucose, ADPglucose, D-glucose 6-phosphate, and D-glucose 2-phosphate cause rate reductions of less than 10%. The origin of the relatively strong binding of the cyclic ester to the phosphorylase, and its possible biological significance are discussed.


The degradation of articular collagen by neutrophil proteinases. The action of the serine proteinase (EC 3.4.21--)of human neutrophil leucocytes, elastase and cathepsin G, on cartilage and tendon was investigated. With cartilage, both enzymes first degraded the proteoglycan, then solubilized collagen by an attack on the terminal peptides, destroying the inter- and intramolecular cross-links. There was little degradation of the helical region of the type II collagen. Elastase also solubilized type I collagen from tendon, though this was less susceptible than cartilage collagen, and attacked the terminal peptides and perhaps the helical region of type I skin collagen in solution. Cathepsin G had little or no effect on type I collagen of skin or tendon. Since massive infiltration of joint tissues by neutrophil leucocytes is a prominent feature of inflammatory joint disease, it may well be that elastase and cathepsin G make a significant contribution to the tissue damage that occurs.


The inhibition of rat liver chromatin protease by congeners of the phenylboronic acids. A group of arylalkylboronic acids were synthesized in order to investigate the inhibitory potential of these compounds for rat liver chromatin protease (EC 3.4--). The effect of side chain length, side chain substitution and aromatic substitution on proteolytic activity in chromatin dissociated in salt and urea was assayed. It was determined that a side chain length two carbons long provided the greatest inhibitory effect with complete inhibition attainable at 20 mM concentration of phenylethylboronic acid. Aryl substitution in the ortho position proved to be the most potent structural change with complete inhibition attained by 1 mM concentration of 0-methylphenylethylboronic acid. The binding of these two inhibitors proved to be reversible.


Preparation and properties of trypsin and chymotrysin coupled covalently to poly (N-vinylpyrrolidone). Water-soluble poly(N-vinylpyrrolidone) of molecular weight 10 000 was modified by hydrolysis of 5% of the gamma-lactam rings, and formation of the N-hydroxysuccinimidester. This activated polymer was bound covalently to trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) to complexes of a molecular weight of about 150 000. The bound enzymes showed an increase in stability towards autolysis. Towards small molecular weight substrates the trypsin-poly (N-vinylpyrrolidone) conjugate showed an increase in specific activity of 1: 1.6, whereas the activity towards larger substrate was found to be only 20%. Chymotrypsin-poly(N-vinylpyrrolidone) showed decreased activity against small (50%) and large (7%) molecular weight substrates.


Purification and characterization of aldolase from human erythrocytes. A procedure has been developed for the purification of human erythrocyte aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.1.3). The process involves a specific substrate elution of the enzyme from phosphocellulose followed by a reverse ammonium sulfate fractionation. The preparation has been shown to be homogeneous by analytical ultracentrifugation, thin-layer electrophoresis, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme exhibits a specific activity of 16 I.U./mg protein, a Km of 7.1-10(-6) M for fructose 1,6-bisphosphate, and a substrate specificity (Fru-1,6-P2/Fru-1-P) of 40. The native protein in a tetramer of 158 000 molecular weight possessing identical or nearly identical subunits, an isoelectric point of 8.9, a diffusion coefficient of 4.68-10(-7) cm2/s, and a molecular radius of 4.56 nm. The study shows the enzyme to be a type A aldolase resembling other muscle forms in chemical and physical properties as well as amino acid composition.


Study on the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. I. Role of structural components of adenosylcobalamin the formation of the active site of glycerol dehydratase. A new method of partial chemical synthesis of adenosylcobalamin (Co alpha-[alpha-5,6-diemethylbenzimidazolyl)]-Co beta-adenosylcobamide, AdoCbl) analogs has been developed. A series of derivatives of AdoCbl modified in the nucleoside and nucleotide ligands and corrin macrocycle have been obtained. The interaction of AdoCl analogs with glycerol dehydratase (EC 4.2.1.30) from Aerobacter aerogenes has been investigated. It has been shown that the nucleoside ligand of AdoCbl provides no essential contribution to the binding of apoenzyme but the preservation of the exact structure of the 1-N and 2-C positions of adenine appears essential for the catalysis. The coordination bond between the Co and nucleotide ligand of AdoCl does not play a decisive role in glycerol dehydratase activity. To form the active site of the glycerol dehydrates, the nucleotide in the AdoCbl structure is essential since nucleotide elimination results in a 100-fold increase of Ki for the corresponding analog. In the binding of AdoCbl with apoenzyme, the main role belongs to the corrin macrocycle, in which the e-propionamide group is significant for binding with apoenzyme, but presumably not essential for catalysis.


Study of the mechanism of action of adenosylcobalamindependent glycerol dehydratase from Aerobacter aerogenes. II. The inactivation kinetics of glycerol dehydratase complexes with adenosylobalamin and its analogs. The inactivation kinetics of vacterial glycerol dehydratase (EC 4.2.1.30) in the course of its reaction with adenosylcobalamin (AdoCbl) and its analogs were investigated. It was shown that the inactivation rate of apoenzyme complexes with AdoCbl analogs is determined by the nature of the analogs employed and probably by the rate of their conversion into hydroxycobalamins. A possible inactivation mechanism of glycerol dehydratase is discussed.


Rate equations and simulation curves for enzymatic reactions which utilize lipids as substrates. I. Interaction of enzymes with the monomers and micelles of soluble, amphiphilic lipids. Theoretical aspects of the kinetics of interaction of enzymes with lipid substrates are presented. Rate equations were written and used to simulate v versus S curves for interaction of enzymes with "monomers" (i.e. a molecular solution) or micelles (aggregated form) of the "soluble", amphiphilic lipids. The rate equations were written assuming separate kinetic parameters for the interaction of the enzyme with these two forms. Although the rate equations are based on the kinetic theory of Michaelis and Menten, most of the simulated v vs. S curves were not hyperbolic. A procedure is suggested for determining the kinetic parameters with the aid of a graphic method.


Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.


Wax ester synthesis in the mouse preputial gland tumour. The microsomal fraction from the mouse preputial gland tumour contains an acyltransferase which catalyzes the synthesis of wax esters. The enzyme is inhibited by moderate concentrations of free fatty acids (40 muM or more) but the inhibition is relieved by the addition of bovine serum albumin. The specific activity of the enzyme increases markedly between the 20th and 30th days of tumor growth. A number of other lipid synthesizing enzymes show similar trends for specific activity as related to tumour age.


Purification and characterization of rat liver microsomal monoacylglycerol lipase in comparison to the other esterases. Monoacylglycerol lipase was separated from triacylglycerol lipase and two kinds of esterase in the microsomes by heparin treatment and DEAE-cellulose column chromatography. Monoacylglycerol lipase was purified about 1200-fold by DEAE-cellulose, hydroxylapatite, SP-Sephadex and Sephadex G-100 column chromatography from rat liver whole homogenate. The purified enzyme showed a single protein band by sodium dodecyl sul fate gel electrophoresis. The molecular weight was calculated to be approx. 62 000 by gel filtration on Sephadex G-200 and sodium dodecyl sulfate gel electrophoresis. The enzyme had an isoelectric point of 6.80 and pH optimum of 8.5. The enzyme maximally hydrolyzed long chain monoacylglycerol such as monomyristoylglycerol and hydrolyzed 1(3)- and 2-monoacylglycerol at equal rates and showed a little hydrolytic activity on short chain triacylglycerol such as tributyrylglycerol, but did not hydrolyze long chain triacylglycerol. The enzyme had different Km and V in comparison with the esterase fro various short chain triacylglycerols and long chain monoacylglycerols. Moreover, monoacylglycerol lipase differed immunologically from two kinds of microsomal esterase. Diisopropyl fluorophosphate inhibited the enzyme activity completely.


Rate equations and simulation curves for enzymatic reactions which utilize lipids as substrates. II. Effect of adsorption of the substrate or enzyme on the steady-state kinetics. Theoretical aspects of the kinetics of interaction of enzymes with lipid substrates are presented. Rate equations were written and used to simulate v versus S curves for the following cases: (a) The substrate is adsorbed onto non-catalytic sites of the enzyme or to other proteins accompanying the enzyme. (b) The enzyme is adsorbed, via non-catalytic sites to aggregated forms of the substrate. (c) The substrate is adsorbed onto an externally added protein such as albumin. Although all rate equations are based on the Michaelis-Menten kinetic theory, most of the simulated v vs. S curves were not hyperbolic and some of the v vs. E curves not linear.


Rat heart in culture as a tool to elucidate the cellular origin of lipoprotein lipase. Lipoprotein lipase was determined in 5-day old cell cultures derived from hearts of newborn rats. With the help of the preplating method the cells were subdivided into cultures containing predominantly cardiac myocytes and into those composed mainly of mesenchymal cells. Lipoprotein lipase activity, associated with the mesenchymal cells was ten times higher than the activity found in the cultures containing mainly the myogenic cells. It is suggested that the mesenchymal cells are the progenitors of lipoprotein lipase in rat heart.


Acetate as a carbon source for lipid synthesis in cultured cells. Lipid synthesis from acetate and glucose has been studied in fibroblast cultures grown in lipid free medium containing various concentrations of acetate and glucose. Their role in lipid synthesis was evaluated by comparing the specific activities of total cell lipid, and sterol and fatty acid subfractions, to that of the carbon source in the medium. When glucose was present at 1000 microgram/ml (5.6 mM) and acetate at 100 microgram/ml (1.2 mM), the majority of the total cell lipid and subfractions were derived from glucose. As acetate concentrations increased, lipid synthesis from acetate increased, and glucose declined as a carbon source; in cells grown in the presence of 1000 microgram/ml (12 mM) acetate more of the lipid was derived from acetate than from glucose. When acetate was present at 1000 microgram/ml (12 mM) and glucose was varied from 1000 to 200 microgram/ml (5.6 to 1.1 mM), the relative amount of lipid synthesis from acetate was further increased. The data indicate that acetate can serve as a significant carbon source for lipid synthesis in cultured fibroblasts when it is presented to the cells at high extracellular concentrations relative to glucose.


Substrate specificity in plasmalogen biosynthesis. Desaturation of 1-O-hexadecyloxyethyl-2-acylglycero-3-phosphoethanolamine in developing rat brain. 1-O-[1'-14C]Hexadecyloxyethyl rac-glycerol was administered to 18-day-old rats by intracerebral injection, and incorporation of radioactivity into the brain lipids was determined after 6, 24 and 48 h. Some of the substrate was catabolized by oxidative cleavage of either of the two ether bonds. Cleavage in the hexadecyloxyethyl moiety yielded labeled palmitic acid, whereas oxidative cleaveage of the glycol glycerol ether bond produced O-hexadecyl glycolic acid. The substrate was also incorporated as such into both ethanolamine and choline phospholipids. Evidence is presented for the desaturation by rat brain of 1-O-hexadecyloxyethyl-2-acyl-sn-glycero-3-phosphoethanolamine to the plasmalogen analogue, while the corresponding choline phospholipid was not desaturated.


Diffusion coefficients of single bile salt and bile salt-mixed lipid micelles in aqueous solution measured by quasielastic laser light scattering. Diffusion coefficients of single bile salt micelles and mixed binary micelles consisting of single bile salts and phosphatidylcholine in the molar ratio 3 : 1 have been measured using quasielastic laser light scattering spectroscopy. Among single systems, marked concentration dependence of the diffusion coefficient was observed only in those containing sodium taurodeoxycholate. Micellar size correlates with bile salt molecular structure in a manner similar to that observed by earlier workers with some minor differences to towards a more consistent ordering. Phosphatidylcholine produces small increases in micellar size in most systems depending on bile salt structure, and seems most plausibly interpreted as simple swelling of the single species micelles. In some systems, when phosphatidylcholine is added, size or shape changes are indicated. Diffusion coefficients were also studied in the ternary systems bile salt-phosphatidylcholine-cholesterol which were saturated and metastably super-saturated with respect to cholesterol. The Stokes radii in both systems were the same within experimental error, 35 A. Our results indicate that intermicellar interactions significantly contributed to the measured diffusion coefficients in the ternary systems as well as in the single sodium taurodeoxycholate and binary sodium taurodeoxycholate-phosphatidylcholine systems in the concentration range studied.


Production and effects of 7 alpha-hydroxytestosterone on testosterone and dihydrotestosterone metabolism in rat testis. 1. Testicular 7 alpha-hydroxylation of testerone was assayed in cell extracts of rats between 12 and 79 days of age. Maximal 7 alpha-hydroxylase activity was observed about 60 days, while insignificant activity was obtained prior to 42 days of age. 2. 7 alpha-Hydroxytestosterone, a major metabolite of testosterone in mature rat testis, inhibited 5 alpha-reduction of testosterone in cell extracts of mature but not of immature rat testis. 3. Maximal testicular activity of 3 beta-hydroxysteroid dehydrogenase using dihydrotestosterone as substrate was obtained in the presence of NAD, while maximal 3 alpha-hydroxysteroid dehydrogenase activity was observed with NADP. Both enzyme activites were reversible. 4. Sensitivity toward testosterone inhibition of 3-hydroxysteroid dehydrogenase varied greatly with stage of testis development being highest at 25-27 days of age. In contrast to testosterone, 7 alpha-hydroxytestosterone was an inhibitor of 3 alpha-hydroxysteroid dehydrogenase only. In the mature rat testis 7 alpha-hydroxytestosterone may be a naturally occurring inhibitor of dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol formation.


Hepatic secretion and turnover of serum phosphatidylcholine-cholesterol acyltransferase in male and female rats. The activity of serum phosphatidylcholine-cholesterol acyltransferase (LCAT), output of the enzyme by the perfused rat liver, and the effect of pretreatment with colchicine on LCAT were studied in male and female rats. It was observed that: 1. Serum LCAT activity in the female exceeded that of the male in fasted animals, whereas in fed animals, LCAT activity was higher in the male than the female. With both sexes, however, serum LCAT activity in fed animals was greater than that in fasted animals. Data are presented which suggest that the observed sex differences were due to concentration and/or composition of the substrate rather than to differences in the serum concentration of the enzyme. 2. The release of LCAT by perfused livers from fasted female rats exceeded that of the male animals. The output of LCAT was inhibited by pretreatment (male) with colchicine, which suggests that hepatic secretion of LCAT is dependent on vesicular transport. 3. The decay of serum LCAT activity in vivo following injection of colchicine was more rapid in fasted female rats than in male animals. These observations lead us to postulate that the turnover rate of LCAT is higher in female rats than in male animals.


Rat urinary metabolites of [9,10-methylene-14C] sterculic acid. 1. The metabolism of [9,10-methylene-14C] sterculic acid was studied in corn oil and Stercula foetida oil fed rats. The majority of the radioactivity was excreted into the urine as short chain dicarboxylic acids. The main urinary metabolites were cis-3,4-methylene adipic acid, cis-3,4-methylene suberic acid, trans-3,4-methylene adipic acid, cis-3,4-methylene pimelic acid, and cis-3,4-methylene azelic acid. 2. Formation of these urinary metabolites requires alpha-, beta-, and omega-oxidation plus reduction of the cyclopropene ring to a cyclopropane ring. Sterculic acid must be transported through both mitochondrial and microsomal systems. 3. Other non-radioactive urinary compounds were also identified. A proposed pathway for the metabolism of sterculic acid and possible detrimental effects caused by these metabolites is discussed.


Time-dependent, irreversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by the antibiotic citrinin. The inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by citrinin, an antibiotic, has been studied. The inhibition was the mixed type with respect to 3-hydroxy-3-methylglutaryl-CoA and non-competitive with respect to NADPH. When the enzyme was preincubated with citrinin prior to enzyme assay, however, it caused a time-dependent, irreversible inhibition, possibly by binding to a site distinct from the active center on the enzyme protein.


The structure of the menaquinones with a tetrahydrogenated isoprenoid side-chain. Menaquinones with a tetrahydrogenated isoprenoid side-chain of Oerskovia turbata and Brevibacterium lipolyticum were cyclized to the chromenyl acetate derivatives, which were then submitted to ozonolysis, followed by reduction with dimethylsulfide. The mass-spectrometric analyses of the ozonolysis products revealed the ion peaks at m/e 464 (M+), 449, 422, 407, 267 and 225. These results suggest that the two saturated double bonds are located continuously in the second and third units of the chain starting from the quinone ring, and the menaquinones are designated as 2-methyl-3-II,III-tetrahydromultiprenyl-1,4-naphthoquinone.


Side-chain cleavage P-450 from bovine adrenocortical mitochondria. Reconstitution of enzyme activity. The subunit structure of the cytochrome P-450 from bovine adrenocortical mitochondria responsible for the conversion of cholesterol to pregnenolone (side-chain cleavage) has been studied. Isoelectric focusing in 6 M urea reveals two fractions of identical amino acid composition which differ in apparent isoelectric points and in phospholipid content: fraction SI shows 0.6-1.8 nmol phospholipid per 53 000 daltons and pI approx. 4.0; SII shows 6.6-8.9 nmol phospholipid per 53 000 daltons and pI approx. 7.0. SII can be made to behave on isoelectric focusing like SI by removal of phospholipid and SI like SII when the extracted phospholipid is added to the protein (SI). Enzymatic activity can be restored to SII by addition of heme and to SI by addition of heme together with the phospholipid extracted from P-450 from the fractions SI and SII. This phospholipid contains at least four classes of phospholipid of which two have been tentatively identified as phosphatidylcholine and phosphatidylethanolamine. A variety of phospholipids from commercial sources do not permit reconstitution of enzyme activity. Evidence is presented to show that minor contaminants seen on polyacrylamide SDS gels are not essential for enzyme activity nor do they appear greatly to influence enzymatic activity. The possible role of phospholipid in reconstituting cytochrome P-450 activity is considered.


The role of subunits in tubulin. I. Renaturation of tubulin after treatment with urea and sodium dodecyl sulfate. Bovine brain tubulin treated with urea and SDS partially recovered colchicine binding capacity after removal of SDS in the presence of urea followed by stepwise dialysis against urea solutions of lower concentrations. Tubulin subjected to the above procedure showed the same circular dichroism spectrum between 215 and 260 nm as the native one but showed a slightly different fluorescence spectrum when excited at 290 nm.


Preparation and properties of side-chain cleavage cytochrome P-450 from bovine adrenal cortex by affinity chromatography with pregnenolone as ligand. A method is described for preparing cytochrome P-450 (side-chain cleavage) from bovine adrenocortical mitochondria, by affinity chromatography on pregnenolong-Sepharose beads. The cytochrome P-450 appears in two fractions, a large form of heterogeneous molecular weight (large P-450) and a form of molecular weight 850 000 composed of 16 apparently identical subunits (molecular weight 52 000-53 000); this form is referred to as protein 16. Electrophoresis on polyacrylamide gel yields one main band and two minor bands; the appearance of the gels is identical whether the starting material is large P-450 or protein 16 or protein 16 prepared by an entirely different method. Yields of protein 16 can be increased by rechromatography on pregnenolone-Sepharose of large P-450 made 0.1 mM in NADPH. Large P-450 shows greater than 10 heme groups per 16 subunits and is less active enzymatically than protein 16. Chromatography on Sepharose and analytical ultracentrifugation show that large P-450 is heterogeneous with respect to molecular weight. Protein 16 shows a heme content of 8 nmol/mg protein and for both large P-450 and protein 16 heme content by CO-difference spectroscopy is in agreement with values by pyridine hemochromogen. This method of preparing P-450 is convenient and both large P-450 and protein 16 are highly purified.


Comparison of structural glycoproteins from mucus of different sources. The main structural glycoprotein derived from three mammalian mucociliary epithelial secretions (bovine cervical, human lung and human middle ear mucus) were compared. The glycoprotein was purified on a cesium chloride density gradient after prior cleavage of disulphide bonds. Marked similarities were seen for the glycoproteins in their sugar and amino acid composition, in electrophoretic mobility, in sedimentation rate and in their banding densities in a cesium chloride density gradient. The molecular weight of these materials was approximately 0.6-10(6). The similarity noted for these materials occurred despite two of them (lung and middle ear) being derived from pathological sources. The glycoprotein derived from an amphibian mucociliary epithelial secretion (frog palatal mucus) was different; it banded at a lower buoyant density and had a lower sugar content. These findings are discussed in terms of the rheological properties and physiological transport role of mucus on ciliated epithelia. It is suggested that reported differences in the properties of mammalian secretions from different sources is due to differences in the extent of crosslinking between glycoprotein units rather than to chemical or structural differences. In the case of the frog its very different composition may be in keeping with its rather different rheological properties.


Location of the globular region in chicken erythrocyte histone H5. Chicken erythrocyte histone H5 has been cleaved by acetic acid hydrolysis at the two aspartic acid residues 65 and 99 and the 4 peptides (1-65), (66-185) (1-99) (100-185) recovered in a pure form. 270 MHz magnetic resonance and circular dichroic studies show that the two C-terminal peptides are unable to form secondary or tertiary structure. The N-terminal peptides however, form both secondary and tertiary structure. In particular, the peptide (1-99) at high ionic strength possesses a similar number of helical residues to intact histone H5 and also had a closely related nuclear magnetic resonance spectrum. It is concluded that the peptide (1-99) contains most, but not quite all of the residues that are included in the globular segment of histone H5.


Actin from the nematode, Caenorhabditis elegans, is a single electrofocusing species. We have prepared actin from wild type Caenorhabditis elegans animals by three procedures: a purification dependent on the ability of actin to form F-actin, affinity chromatography which preferentially binds G-actin, and co-precipitation of an actin-myosin complex by antimyosin antibodies. Each preparation yields a single electrofucsing species of actin. Comparison of actin from C. elegans embryos and animals reveals that embryos also have the same single electrofocusing species of actin.


Studies on prolactin. 40. Chemical modification of tyrosine residues in the ovine hormone. The seven tyrosine residues in ovine prolactin have been modified, in separate experiments, with tetranitromethane and iodine at 0 degrees C. Both the nitration and iodination studies indicate Tyr-44 to be the most reactive; this residue was converted almost completely to the dinitro and diiodo derivative under the reaction conditions employed. All other reactive tyrosine residues formed the monosubstituted derivative. The relative chemical reactivity of the seven tyrosine residues can be described as follows: Tyr-44 and Tyr-195 are highly reactive, Tyr-147, Tyr-169 and Tyr-185 are partially reactive, with Try-96 and Tyr-28 being relatively unreactive under the conditions employed. The various derivatives have been characterized by exclusion chromatography, circular dichroism, rate of tryptic digestion, and biological activity. The modification of Tyr-44 possibly produces a conformational change which is observed on Sephadex G-100 as an equilibrium between two monomeric forms eluting at slightly different positions. These two forms also appear to display different side-chain CD spectra. Nitration or limited iodination does not appear to decrease the biological activity of the hormone, as measured in the pigeon crop-sac assay, although more extensive iodination does produce a significant loss in potency.


Isolation and characterization of multiple hamster hemoglobins. Multiple hemoglobins were found in the peripheral blood of hamsters when separated by analytical isoelectric focusing in polyacrylamide gel between pH 6.0 and pH 8.0. Five of these hemoglobins (I-V) were present in sufficient quantities for isolation by preparative isoelectric focusing and subsequent characterization. Cellulose acetate electrophoresis of the isolated hemoglobins resulted in producing as many as six different globin chains. The five hemoglobins were further characterized by their amino acid composition, N-terminal amino acid residue, and molecular weight.


Isolation, characterization and partial amino acid sequence of a phosphorylated polypeptide (E4) from bovine embryonic dental enamel. A phosphorylated polypeptide (E4) of molecular weight 5000-6000, has been isolated from bovine embryonic enamel by Bio-Gel P-10 gel filtration and DE-52 ion-exchange chromatography. The peptide contains three serine residues all of which are phosphorylated. All three O-phosphoserine residues are in glutamic acid-O-phosphoserine-tyrosine sequences that are distributed relatively evenly along the polypeptide chain. Although it was not possible to sequence the entire polypeptide chain directly by automatic peptide sequencing, a partial sequence and peptide map was constructed on the basis of the sequence and composition of peptides derived by cyanogen bromide, trypsin and chymotrypsin digestion. The presence of glutamic acid, tyrosine and leucine adjacent to and near the O-phosphoserine residues may be important in calcium binding and in mineralization.


Sequence analysis of porcine gut GLI-1.

Sequence analysis of porcine gut GLI-1. A protein from porcine gut with 100 amino acid residues (porcine gut GLI-1) and having glucagon-like immunoreactivity has been characterized by partial sequences. The sequence of the C-terminal amino acid residues is -Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala and includes the C-terminal amino acid residue sequence (-Met-Asn-Thr) of porcine glucagon. Evidence is presented that the glucagon sequence -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- is found in the gut GLI-1 as well. The data support the theory that gut GLI-1 contains the full glucagon sequence and that gut GLI-1 and glucagon are formed from a common precursor.


The myoglobin of the Cape fox (Vulpes chama). Tryptic, peptic and chymotryptic peptides from the myoglobin of the Cape fox (Vulpes chama) have been aligned with the sequence of the myoglobin of the domestic dog. No amino acid difference was found. Seven carnivore myoglobins now known are compared amongst themselves and it is noted that though the differences between them are found in many different parts of the myoglobin molecule, there seems to be a higher incidence along the loop formed by helix G, the interhelical region GH and the helix H.


Regulation of hydrogen peroxide in eye humors. Effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye. Activities of catalase (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) and GSH peroxidase (GSH: H202 oxidoreductase, EC 1.11.1.9) have been measured in iris, ciliary body, retina, corneal epithelium, corneal endothelium, lens capsule-epithelium and decapsulated lens. 3-Amino-1H-1,2,4-triazole is a specific inhibitor of catalase and a potent cataractogenic agent. We observed marked inhibition of catalase activity in these tissues 1--6 h after the administration of a single intravenous dose of 1 g 3-aminotriazole per kg body weight in rabbit. This was associated with a 2--3-fold increase in the H2O2 concentrations of aqueous humor and vitreous humor. The increased peroxide concentrations were restored to the physiological levels as the catalase activity of eye tissues gradually returned to normal with time after injection. Under the conditions, GSH peroxidase activity of the afore-mentioned eye tissues was unaltered, GSH and protein sulfhydryl of lens were not changed, and ascorbic acid of aqueous humor and vitreous humor was not significantly altered.  Based on these findings our conclusion is that catalase of eye tissues regulates the endogenous H2O2 in eye humors to the physiological level. We speculate that H2O2 may be the triggering factor in cataract induced by 3-aminotriazole.


Ornithine decarboxylase activity during neonatal development of the rat ventral prostate. The relationship between ornithine decarboxylase activity and growth has been examined in ventral prostates from rats aged 2--60 weeks. The incorporation of [125I]iododeoxyuridine in vitro was used to assess DNA-synthetic activity. During 2--7 weeks post-partum ornithine decarboxylase activity/prostrate and the protein content and wet weight increased exponentially. In contrast, the incorporation of [125I]iododeoxyuridine/prostate fell during the first 4 weeks. This decline was reversed at about the time when the systemic concentrations of gonadal androgens start to rise. After 10 weeks post-partum the incorporation of [1252]iododeoxyuridine/prostate and ornithine decarboxylase activity/prostate levelled off, whereas the DNA and protein content of the prostate continued to increase at a slow rate.