[Production of infarction model in dogs by several brain arteries occlusion. Part 1: Production of hemispheric non blood supply model (author's transl)]. Experimental brain infarction in model animals should most ideally be constant in the site of occurrence, be of severity that is controllable by the occlusion time of blood flow and, moreover, be reproducible at a high rate to desired extent and severity. However, among infarction models elaborated in animals so far, only thalamus infarction model in dogs which were reported by us previously have been proven to satisfy almost all of these requirements. In experiments with these model dogs we found that an extensive infarction can be produced at a high frequency in the involved hemisphere by a transient occlusion of blood flow at such intracranial levels as the internal carotid artery, the junction of the anterior cerebral artery and ophthalmic artery, the middle cerebral artery, the origin of the segment A2 of the anterior cerebral artery, and the junction of the posterior communicating artery and posterior cerebral artery. During about one hour following the releasing of the arteries approximately 2 hours occlusion, the development of severe brain edema resulted involving the whole hemisphere on the affected side invariably in all cases. This method for experimental production of brain infarction permits to control the degree of resultant brain edema by changing the duration of occlusion of blood flow, requires simple manipulations only and ensures production of a constant lesion. With these advantage it can be anticipated to prove of great benefit in the study of postischemic brain edema.


Survival of microorganisms and helminth eggs in green pills and cobs processed from a green crop irrigated with sewage. A grass crop which was contaminated with domestic sewage was dried in a hot air drying plant in order to observe the effect of the drying on pathogenic bacteria, virus and parasite eggs. The investigation showed, that pathogenic bacteria and virus were reduced with a factor 10(5)--10(6), and that parasite eggs were reduced with at least a factor 200, which is sufficient to secure that grasis irrigated with sewage and pre-served with hot air drying is used without hygienic risks.


[Diagnosis and treatment of cerebellar hemorrhage: comparison of hypertensive hemorrhage with hemorrhage caused by small angiomas, and CT findings (author's transl)]. In spontaneous cerebellar hemorrhage emergency surgical intervention is often life-saving. Clinical features and the operative results of hypertensive cerebellar hemorrhage (18 cases) were compared with those of hemorrhage caused by small angiomas (7 cases). Hypertensive hemorrhage occured most frequently in the seventh decades. Two thirds of the patients developed brainstem compression syndrome within a week from onset. One third remained awake or drowsy throughout their clinical course. Surgical removal of a hematoma was carried out in 13 patients with four deaths. Of note, two comatose patients regained consciousness after surgery, and were discharged with residual ataxia. Rupture of a small angioma occurred in younger patients. Their clinical course was sub-acute or chronic associated with focal cerebellar dysfunction. All seven surgically treated patients subsequently regained independent function. CT findings have been found helpful not only for diagnosis but also in defining appropriate therapy. Hematomas larger than 3 cm in diameter produced signs of rapidly progressing compression of the brainstem. Thereby, regardless of the cause of bleeding, emergency removal of a clot is indicated even in awake patients. Hematomas of 2 to 3 cm produced brainstem compression or prolonged cerebellar dysfunction, and occasionally require surgical decompression. Hematomas smaller than 2 cm can be managed conservatively, since they were absorbed spontaneously in three weeks without residual functional disturbances. However, in case of a young patient exploration should be performed for a probable "cryptic" angioma.


[The correlation between autoregressive power spectrum of EEG and computerized tomography in tuberous sclerosis (author's transl)]. The autoregressive power spectrum and their component analyses of 36 electroencephalograms of 9 patients with tuberous sclerosis and 21 healthy children were studied. The data were recorded on the analog tapes, and the 20 second artifact free segment of records was digitized at 50 samples/sec. The autoregressive power spectrum and their component were calculated by the methods of Sato (1976) with the minicomputer PDP 11/40. The component consisted of the first and the second order elementary processes. The former showed a transient nonscillatory delta wave, whereas the latter showed damped oscillatory waves of delta, theta, alpha and beta rhythms in the EEG. The characteristics of these component rhythms in the EEG were given by the frequency, the time constant of the nonoscillatory delta, the damping time of the oscillatory component waves (time constant of the envelope of the damped oscillation), the mutual information amounts, etc. Thus, the correlation between these characteristics of component and CT-scan in tuberous sclerosis were examined. The results were as follows: 1. Compared with the characteristics of EEG in normal children, the mutual information amount, the damping time and/3r time constant showed significantly lower value in alpha rhythms of frontal-, central-regions and in theta, delta rhythms of occipital region in the patient with tuberous sclerosis. 2. Multiple subependymal high density areas were found in all of these patients on CT. EMI-number of these high density areas were less than that of calcification in younger children below the age of 3 years, but in older children were equivalent to that of calcification. 3. The correlation between the number of subependymal nodule on CT and the mutual information amount in the EEG showed significantly the negative coefficient in theta, delta rhythms of O1 region in older age group of patients. 4. The correlation between EMI-number of these subependymal high density areas and the mutual information amount in the EEG showed significantly thenegative coefficient in theta & delta rhythms of O1 region in all cases of patients. 5. It is considered that the multiple subependymal nodules may influence the background activities of the central and the occipital regions in the EEG of tuberous sclerosis.


Ketamine block of bronchospasm in experimental canine asthma. Experimental asthma was induced in dogs previously sensitized to ascaris antigen by ventilation with ascaris antigen, in an aerosol, for 10 min. Before the administration of antigen, there was no significant difference in pulmonary airways resistance (RL) during thiopentone and ketamine anaesthesia. In dogs anaesthetized with thiopentone, RL increased significantly from a pre-antigen control of 0.36 +/- 0.13 (mean +/- SEM) kPa litre-1 s to 1.56 +/- 0.38 at 5 min after administration of antigen. In dogs anaesthetized with ketamine, RL before (0.30 +/- 0.10) and 5 min after antigen (0.47 +/- 0.18) was not significantly different. Beta adrenergic blockade with propranolol abolished the protective effect of ketamine so that there was no significant difference in the maximal increase (5 min after antigen) in RL in dogs anaesthetized with ketamine (2.92 +/- 0.74) or thiopentone (3.28 +/- 1.16). Beta adrenergic blockade also increased pre-antigen RL in both groups (ketamine 0.87 +/- 0.24; thiopentone 0.77 +/- 0.32).


Prolongation by bile salts of the duration of action of a steroidal neuromuscular blocking agent. The influence of bile salts on the duration of action of the steroidal non-depolarizing neuromuscular blocking agent ORG 6368 was investigated in cats. The intravenous administration of ORG 6368 (100 microgram/kg body wt) caused a maximum neuromuscular blockade of 71 +/- 6% with a duration of action of 3.4 +/- 0.1 min. However, intraportal administration of the same dose caused no significant neuromuscular blockade. Following an infusion of dehydrocholate 320 mumol, lasting for 8 min, the magnitude and the duration of action of the neuromuscular blockade produced by ORG 6368 were increased markedly. This effect of the bile salt is possibly a result of inhibition of the hepatic uptake of ORG 6368, thereby retarding its disappearance from the plasma and consequently prolonging the neuromuscular blockade. The neuromuscular blocking effect of intraportally administered gallamine (1 mg/kg body wt) was not influenced significantly by the infusion of dehydrocholate.


Effect of acute hypocapnia on some aspects of renal function in anaesthetized dogs. Hypocapnia was induced in dogs lightly anaesthetized with nitrous oxide and fentanyl. Measurements were made of estimated renal plasma flow (ERPF), glomerular filtration rate, vascular resistance and urine production. During the (short) duration of the experiments hypocapnia was found to be associated with ERPF twice that during normocapnia. Glomerular filtration rate and urine output were increased by hypocapnia in an approximately similar proportion, whilst renal vascular resistance halved. Though some of the experimental animals had a degree of metabolic acidosis this was thought not to have greatly influenced the results.


Cytotoxicity of i.v. anaesthetic agents on the isolated rat hepatocyte. The isolated rat hepatocyte model has been used to assess the hepatotoxicity of a number of i.v. anaesthetic induction agents. Ketamine, Althesin and CCI 12923 (minaxolone) all inhibited gluconeogenesis and urea formation from alanine. There was also a decrease in the cell ATP concentration, and a dose-related increase in leakage of LDH. Of these indices of cell toxicity, gluconeogenesis from alanine was found to be the most sensitive. Fifty per cent inhibition of gluconeogenesis for all three agents occurred in the range 150--300 mumol. The effects of these agents on the isolated hepatocyte may be attributed to a primary impairment of mitochondrial function through a change in the ATP concentration. The plasma concentration of anaesthetic agents measured during their clinical use is at least one order of magnitude less than that required to cause 50% inhibition of gluconeogenesis.


Pharmacokinetics of fentanyl as a possible explanation for recurrence of respiratory depression. The pharmacokinetics of fentanyl are complicated by an additional increase in plasma concentration during the elimination phase of the drug. We have confirmed that fentanyl is excreted in the gastric juice and reabsorbed from the alkaline medium of the small intestine. In addition, the stomach wall in rats has an important storage capacity for fentanyl. A maximum of about 20% of the dose was found in the stomach wall after i.v. injection. In man the resected part of the stomach contained 16% of the dose, 10 min after injection. These observations could be important in explaining the occurrence of respiratory depression in the period after operation.


A screening test for the malignant hyperpyrexia phenotype using suxamethonium-induced contracture of muscle treated with caffeine and its inhibition by dantrolene. Suxamethonium induced a contracture in caffeine pretreated human muscle in vitro. The contracture was significantly greater (P less than 0.001) with MHS muscle compared with muscle from normal subjects. This reaction is now used as an additional screening test for the MHS phenotype. The contracture was prevented by pretreatment with dantrolene.


Halothane hepatitis revisited in women undergoing treatment of carcinoma of the cervix. From 1964 to 1976, 238 patients with carcinoma of the cervix were treated with irradiation and surgery. Their medical records were reviewed retrospectively. Three patients became jaundiced after operation as a result of pigment overload and sepsis. Exposure to halothane did not appear to be a significant aetiological factor.


Effect of maternal position on fetal heart rate during extradural analgesia. Using continuous cardiotocography, the effect of maternal position on fetal heart rate (FHR) was studied during extradural analgesia (EA) with either 50 or 100 mg of etidocaine without adrenaline for labour and vaginal delivery. Of 70 healthy parturients, 39 were supine and 40 were in the lateral position. FHR was normal in all patients during the control period before EA, and remained normal during EA in 53. Transient abnormal patterns occurred in 26 patients, 22 (56%) in the supine group and four (10%) in the lateral group, a significant difference (P less than 0.001). The decrease in arterial pressure (AP) did not differ between the groups, but the frequency of abnormal FHR associated with a small or moderate decrease in AP (30% or less) was greater in the supine (51%) than in the lateral group (3%).


Has droperidol an atropinic effect?

Has droperidol an atropinic effect? The action of droperidol on the tachycardia produced by atropine and on the serum concentration of cholinesterases was observed during balanced anaesthesia. Without atropine, the mean heart rates of patients who received fentanyl plus droperidol were similar. Atropine increased heart rate only in the presence of droperidol (P less than 0.001) (fentanyl v. fentanyl plus droperidol: P less than 0.05). Droperidol inhibited serum cholinesterases (P less than 0.05); this effect was independent of atropine.


Venous sequelae following etomidate.

Venous sequelae following etomidate. Five hundred patients receiving one of three different formulations of etomidate were observed on the 2nd and 3rd days after operation for possible venous sequelae. The total frequency of sequelae was 8% or more, the greatest being with the propylene glycol formulation (23%). Formulation of the drug and total dose employed were the most important factors influencing venous sequelae, which were not related to pain on injection.


Non-depolarizing neuromuscular blockers and the eye: a study of intraocular pressure. Pancuronium versus alcuronium. Intraocular pressure remained stable when either pancuronium or alcuronium was used to produce muscle relaxation, suggesting that alcuronium may be a reasonable alternative to pancuronium for routine surgery of the open eye, particularly in elderly patients with limited cardiac reserve.


Hepatitis following thiopentone. A case report. A patient developed pyrexia and jaundice following repeated anaesthesia. The evidence suggests that thiopentone was the causative agent. This incident emphasizes the need for caution in associating jaundice and hepatitis with halothane in patients who have received thiopentone and halothane.


Cimetidine: effect on pancreatic and biliary function in man. 1 Pancreatic and biliary function were studied in eight patients taking oral cimetidine (1.6 g/day for 6 weeks) for endoscopically confirmed active peptic ulcer disease. 2 Postprandial jejunal tryptic activity and bile acid concentration were unchanged during cimetidine given for 1 and 6 weeks. 3 Postprandial gallbladder emptying was also unchanged. 4 Direct examination of postprandial upper jejunal aspirate for deconjugated bile acids was negative in all patients; but the more sensitive [14C]-glycocholate bile acid breath test showed a significant increase in breath 14CO2, indicating increased bacterial deconjugation of bile acids in the gastrointestinal tract.


Biliary excretion of erythromycin after parenteral administration. 1 The biliary excretion of erythromycin has been studied following parenteral administration in 23 patients. 2 Mean bile levels of the drug were approximately ten times higher than corresponding serum concentrations 1 h after i.v. (erythromycin lactobionate) and i.m. (erythromycin ethylsuccinate) injection. 3 Thus, unlike many antimicrobial agents, these compounds are well concentrated in the bile.


A gastroscopic and pharmacological study of the disintegration time and absorption of pivampicillin capsules and tablets. 1 An in vitro investigation showed that pivampicillin tablets disintegrated more rapidly than pivampicillin capsules. This result was demonstrated and confirmed by gastroscopy in a cross-over study in healthy volunteers. 2 There were no differences in serum levels of ampicillin obtained with the two preparations, but compared with non-gastroscoped volunteers, there was delay of 0.5--1.5 h in the appearance of peak serum ampicillin concentrations after gastroscopy. 3 Half of the volunteers receiving pivampicillin capsules developed hyperaemia, interstitial bleeding or erosions of the gastric mucous membrane. No such reactions were seen after pivampicillin tablets. 4 In one volunteer, a pivampicillin capsule was trapped in a not previously noticed hiatus hernia and local changes and pain occurred.


Pharmacokinetics and pharmacodynamics of warfarin at steady state. 1 The relationship between warfarin dose, total and free plasma warfarin concentration, and anticoagulant effect was examined at several steady-state levels in fifteen patients during withdrawal of warfarin therapy. 2 Total plasma clearance was significantly correlated with the free fraction in plasma (r=0.955). 3 There was an age related decline in the dose of warfarin, and in the total and free plasma warfarin concentrations required to produce the same anticoagulant effect. However, neither total nor free plasma warfarin clearances varied with age. 4 Individual patients' log concentration-effect relationships were linear above a prothrombin ratio of 1.2 and there was a significant correlation (r=-0.586) between the slope and the free fraction of warfarin in plasma. It is suggested that plasma protein binding may reflect the interaction between warfarin and its effector site in the hepatocyte.


The pressor actions of noradrenaline, angiotensin II and saralasin in chronic autonomic failure treated with fludrocortisone. 1 Treatment of postural hypotension due to chronic autonomic failure with fludrocortisone increased the pressor sensitivity to intravenous noradrenaline. Fludrocortisone increased the blood pressure in the standing but not the lying position. These effects of fludrocortisone may be the result of increased sensitivity of vascular receptors to noradrenaline. 2 The pressor action of angiotensin II, to which patients were supersensitive, may have involved the stimulation of alpha-adrenoceptors since it was partially antagonised by phentolamine. 3 Saralasin had a marked, paradoxical, pressor effect. This may have been mediated by vascular alpha-adrenoceptors because log dose-response curves of saralasin-induced increases in systolic pressure were shifted to the right in a parallel fashion after phentolamine. 4 Fludrocortisone treatment increased the pressor sensitivity to intravenous saralasin but not to angiotensin-II.


A pharmacokinetic comparison of two sustained-release oral procainamide preparations. 1 The pharmacokinetics of two different sustained-release oral procainamide preparations were studied in ten hospital patients with normal blood ureas and no clinical evidence of heart failure. Each patient received either one or other preparation at 12 hourly intervals for four doses. Frequent blood sampling enabled close monitoring of blood levels. 2 Results showed that both preparations were essentially similar in their pharmacokinetics. Both effectively double the half-life of conventional oral procainamide to 6.5 h and are suitable as prophylactic preparations. One patient developed toxic levels, thought to be related to her metabolic status of being a very slow acetylator. To avoid toxicity pre-therapy assessment of a patient's cardiac and renal function and acetylator status is advised.


A new theory of carcinogenesis.

A new theory of carcinogenesis. Although many carcinogens are mutagens, there is no direct evidence that the cancer-cell phenotype is the result of gene mutation. Transplantation experiments have strongly indicated that malignant cells can arise or revert to the normal phenotype in the absence of mutation. It is suggested that damage to DNA followed by repair triggers the epigenetic changes in gene expression which are responsible for malignancy. We previously proposed that methylation of specific DNA sequences adjacent to structural genes determines whether or not transcription will occur. Specific methylases are required for the switching on of genes and for the stable maintenance of the methylated state, which provides a basis for the control of gene expression in differentiated cells. It is now seen that damage to DNA followed by repair, just before or just after DNA replication, can lead to the loss of methyl groups. This can induce a switch in gene activity which is heritable, but potentially reversible. The known large difference in the probability of malignant transformation in cells of rodents and large mammals is hard to explain if mutation is responsible. On the other hand, this new theory provides an explanation for this difference, since the probability of epigenetic changes in gene activity will depend on the activity of methylating enzymes and the rate of excision repair. The theory is supported by the evidence that excision repair is more efficient in cultured fibroblasts from large long-lived animals than from small short-lived ones.


Cytotoxic and clastogenic effects of soluble chromium compounds on mammalian cell cultures. The inhibition of cell growth, the reduction of cell survival and the induction of chromosome aberrations and of sister chromatid exchange (SCE) have been determined in cultured hamster cell lines (BHK and CHO) treated with 11 water-soluble compounds of hexavalent and trivalent chromium. All Cr6+ compounds inhibit growth of BHK cells and reduce survival of CHO cells to levels comparable to those obtained only after exposure to 100--1000 times higher Cr3+ concentrations. The cytotoxicity curves obtained with the different Cr6+ compounds are almost overlapping, whereas marked differences of activity are noticeable among Cr3+ compounds. Giant cells are obtained after exposure to Cr6+ and Cr3+ compounds, as shown by the rise of DNA and RNA per cell, and are due to the blockage of the cell cycle without sudden inhibition of macromolecular syntheses. Both Cr6+ and Cr3+ compounds are able to induce chromosome aberrations, whereas Cr3+ is absolutely incapable of inducing SCE, only Cr6+ being active. The frequency of chromosome aberrations is increased about 10-fold after exposure to 1.0 micrograms/ml Cr6+, whereas it is only doubled after treatment with up to 150 micrograms/ml Cr3+. On the other hand, in spite of the sensitivity of CHO cells to the induction of SCE by mitomycin C, the frequency of SCE hardly doubles after exposure to Cr6+ compounds. The present data confirm that Cr6+ compounds are characterized by a marked cytotoxicity and clastogenic action on mammalian cell cultures and show that Cr3+ compounds, though cytotoxic only at extremely high concentrations and not increasing the frequency of SCE, are not completely without cytogenetic effect, as they are able to induce chromosome aberrations.


The spread of breast cancer: importance of the intrathoracic lymphatic route and its relevance to treatment. Detailed necropsies were performed on 26 individuals who had died of disseminated breast carcinoma, to assess the frequency of spread to the lungs, pleura and pericardium, and to determine the likely routes of spread to these sites. Tumour was present in the lung parenchyma in 67% of the lungs examined, in the visceral pleura in 75% and the parietal pleura in 50%. Although even small deposits of pleural tumour were invariably visible to naked-eye examination, lung parenchymal involvement was almost invariably microscopic, despite its frequently extensive distribution. This finding draws attention to the difficulties of clinical staging with respect to lung metastases. Tumour in lymphatics predominated over that in blood vessels in both lung and pleura and this, together with the widespread mediastinal lymphnode infiltration found, suggests that the lymphatic system forms the dominant route of spread of breast carcinoma to the thorax. The possible role of mediastinal lymphatics in the dissemination of breast cancer to bone and liver is also discussed. Our findings suggests that the fields of adjuvant irradiation after primary surgery should include the mediastinal lymphatic network.


Incidence of nasopharyngeal carcinoma in Malaysia, 1968--1977. A record of all known cases of nasopharyngeal carcinoma in Malaysia is complete for 10 years from 1968 to 1977. Special efforts in case-finding were made in the State of Selangor where conditions are optimal. Age-adjusted incidence rates among Chinese males and females were 16.5 and 7.2 per 100,000, among Malay males and females 2.3 and 0.7 and among Indian males, 1.0. There were no significant changes in incidence rates over the 10-year period for sex and ethnic groups, or for Chinese subethnic groups. In Chinese subethnic groups, rates were highest among Cantonese, moderate among Khek and lowest among Hokkien and Teochiu. Standardized incidence ratios using Selangor as the standard population indicate considerable under-reporting in the less urban states of Malaysia, particularly among females. In Selangor, incidence rates were similar for urban and rural residents, but the frequency of cases was higher among Chinese working in industry and living in poor neighbourhoods.


Histopathological changes of the nasal mucosa in active and retired nickel workers. Histological examinations were made on nasal biopsy specimens from the middle turbinate in 318 active and 15 retired nickel workers and in 57 controls, to study the prevalence of nasal carcinoma or possible precancerous mucosal changes in nickel-exposed individuals. The histopathological changes were evaluated according to a point-score scale, and the results were correlated to age, smoking habits, duration and type of nickel exposure and to nickel concentrations in nasal mucosa, plasma and urine. The explanatory values of these factors on the histopathology were estimated by stepwise multiple regression analysis. Two nickel workers from the roasting/smelting department (0.6%), both employed 28 years at the plant, had nasal carcinoma. Epithelial dysplasia was found in about 12% of active and 47% of retired nickel workers. One of the controls, a male carpenter, had dysplasia. These histopathological changes may be precancerous lesions, as they are almost exclusively found in active and retired nickel workers with enhanced risk of nasal carcinoma. Loss of respiratory epithelium and development of squamous epithelium were regarded as unspecific histopathological changes. These changes were seen in all groups, even though in significantly higher incidence in the nickel-exposed groups. Duration of nickel exposure, type of nickel-refining work and tobacco consumption were the independent variable that, taken altogether, had the highest explanatory values for the histopathological changes.


Causes of death of blue-collar workers at a Dublin brewery, 1954--73. The suggested association between high consumption of beer and an increased risk of death from cancer of the colon and rectum was investigated among blue-collar workers at a Dublin brewery, who consume more than average amounts of beer, usually in the form of stout. A study of their mortality between 1954 and 1973 showed that they had as good an expectation of life as all Dublin males, with no increased risk of death from cancer of the oesophagus, pharynx, liver or of cirrhosis of the liver, accidents or suicide, conditions normally associated with the high consumption of alcohol. They had significantly increased risk of death from cancer of the rectum and also from diabetes mellitus. Twenty per cent of the workers, differentiated by their place of work within the brewery, had a much higher risk of death from cancer of the rectum.


Immunity as the predominant factor determining metastasis by murine lymphomas. The metastatic behaviour of the L5178E (non-M) lymphoma and a highly metastatic subline L51787ES (M) were studied in syngeneic DBA2 mice. The non-M tumour rarely metastasizes in intact syngeneic mice, but produces extensive and rapidly lethal metastases when implanted into irradiated recipients. The metastatic behaviour of the M subline is unaffected by irradiation of the host. By conventional transplantation criteria, the non-M tumour is more immunogenic than the M subline. Both tumours, however, produce similar responses in a lymphnode weight-gain assay. Host-cell infiltration of the tumours growing s.c. is much greater in the non-M than the M, the infiltrating cells being Fc-receptor-positive and maturing into macrophages after 2 days in vitro. Although spontaneous in vitro motility of the M cells is much greater than that of the non-M, the metastatic behaviour of the tumours is clearly determined by host immunological responses.


The role of macrophages and polymorphs in the levan-induced inhibition of Lewis lung carcinoma in C57BL mice. High-mol.-wt levan injected locally inhibits the growth of Lewis lung carcinoma in C57BL mice. The inhibition is dependent on the number of tumour cells injected and on the dose of levan. The inhibition decreases tumour incidence and size as well as prolonging survival. The polysaccharide is most effective when injected daily beginning on the day of tumour-cell inoculation. Treatment begun on later dates is less effective. Treatment begun one day before tumour-cell inoculation enhances tumour growth. Histological studies showed that levan induces an intense polymorphonuclear (PMN) reaction followed by accumulation of vacuolated, levan-laden macrophages. Both PMN and activated macrophages seemed to have an inhibitory effect upon the growth of the tumour. The effector role of PMN was not explained by the histological study. Tumour cells in close contact with levan-laden macrophages appeared mostly necrotic. Administration of levan begun one day before tumour-cell inoculation produced a similar reaction, but the infiltrating cells did not appear to approach and damage the tumour cells.


Bovine ocular squamous-cell carcinoma: lymphocyte response to phytohaemagglutinin and tumour antigen. The presence of ocular squamous-cell carcinomas in cattle was associated with significantly lower blastogenic response of peripheral-blood cultures to PHA than that of age-matched control cattle. The difference in blastogenic response was more marked when the external diameter of the tumour exceeded 2 cm. Cattle with aquamous-cell carcinomas had cell-mediated immunity against tumour extracts, as measured by the leucocyte adherence inhibition (LAI) microassay. The LAI reaction against tumour extracts was directly proportional to the general level of cell-mediated immunity as demonstrated by the lymphoproliferative response to PHA.


Investigation of time-gap formulae on the CRE system using mouse tissue as a biological model. The cumulative radiation effect (CRE) is one of several empirical scalar descriptions of biological effect which enable corrections to be made for gaps in radiotherapy treatment. Predictions of this theory were tested using mouse crypt regeneration and mouse skin as biological models. These experimental results are discussed in terms of the dependence of tissue regeneration potential during a gap on the biological effect achieved before the gap, and on gap length. A hypothesis is proposed to reconcile the apparent conflict between the two experiments. While the simple exponential gap formulation of the CRE is seen to be inadequate, insufficient data are available at present to modify it.


Reduced granulocyte-macrophage colony-stimulating activity by mitogen-stimulated lymphocytes from patients with aplastic anaemia. The ability of peripheral blood mononuclear cells from patients with idiopathic aplastic anaemia to provide colony-stimulating activity (CSA) was compared to that of normal controls. CSA was prepared by incubating peripheral blood mononuclear cells with phytohaemagglutinin. The supernatant derived from the latter is known as activated lymphocyte-conditioned medium (ALCM). The CSA of ALCM in eight patients was compared to that of normals in 12 experiments. In all but one instance there was decreased CSA by patient ALCM. Possible implications of these findings are discussed.


Effects of 5-fluorouracil on mouse bone marrow. 5-Fluorouracil (150 mg/kg) was injected intravenously into 11-week-old male mice. Its effects on marrow structure over the following 9 d were assessed using light microscopic examination of semi-thin transverse sections of decalcified humeri. The extravascular compartment became markedly depleted of cells and decreased in size, reaching a minimum on day 5 post-treatment. Proliferating haematopoietic cells disappeared within 2 d. Post-replicative erythrocytic cells underwent extra-vascular phagocytosis by resident macrophages, whilst mature granulocytes continued to pass into the circulation. Repopulation was underway by day 6, and re-expansion of the extravascular compartment by day 7 when megakaryocytes, of larger than normal average size, occupied most of the extravascular compartment. Megakaryocyte dominance gave way over days 8 and 9 as other haematopoietic cells proliferated and the extravascular compartment continued to enlarge. The significance of the results is discussed in relation to marrow micro-circulation, egress of haematopoietic cells into the circulation, and cell proliferation in the marrow. Endotoxin, administered 4 h after 5-fluorouracil, accelerated depletion of the marrow and recovery of haematopoiesis by about 1 d.


Rheological properties of erythrocytes in heterozygous and homozygous beta thalassaemia. The rheological properties of erythrocytes were studied in 12 heterozygous and nine homozygous beta thalassaemia patients, using filtration of erythrocytes through polycarbonate sieves with a pore diameter of 5 micron and viscosity measurements of erythrocyte and ghost suspensions. Erythrocyte deformability of all patients was decreased. In the heterozygous patients moderately diminished flexibility of cells was due to microcytosis of erythrocytes. Rigidity of erythrocytes in homozygous patients was due to altered cell shape, diminished fluidity of haemoglobin, and in splenectomized patients to the presence of inclusion bodies. Membrane flexibility of erythrocytes remained unaltered. We suggest that the decreased fluidity of haemoglobin as well as the presence of inclusion bodies result from the excess of alpha chains in erythrocytes of homozygous patients. The haemoglobin fluidity of erythrocytes of one patient with thalassaemia intermedia was unchanged. The flexibility of these cells was only moderately altered. In four patients who needed transfusions every 1--4 weeks deformability of transfused erythrocytes was likewise decreased when compared to controls. Erythrocyte suspensions obtained from the splenic artery and vein during splenectomy showed decreased flexibility of transfused cells after their passage through the spleen.


Alpha thalassaemia in Sicily: haematological and biosynthetic studies. Eight Sicilian patients with Hb H disease and their families have been studied. The standard haematological tests and the alpha/beta chain synthesis ratios showed significantly different results in the patients with Hb H disease as compared with alpha thalassaemia carriers, except for Hb A2 values. There was no significant difference in the mean RBC, MCV, Hb A2, Hb A1 and Hb F of alpha thalassaemia carriers compared with normal controls. On the contrary significant difference was found between the mean alpha/beta chain synthesis ratio of alpha thalassaemia carriers and that of the normal controls; however, the extensive overlapping of alpha/beta values between these two conditions make this parameter insufficiently discriminant. No correlation was found between MCV, MCH, RBC and alpha/beta chain synthesis ratio in patients with alpha thalassaemia trait, suggesting that the ratio cannot be used to distinguish between carriers of a mild gene ('silent' carrier) and carriers of the more severe alpha thalassaemia gene. A possible genetic model for alpha thalassaemia in Sicily is presented.


Distribution of erythrocyte nucleotides in pyrimidine 5'-nucleotidase deficiency. In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.


Hereditary haemolytic ovalocytosis with defective erythropoiesis. Patients belonging to four families with 'atypical elliptocytosis' have been investigated. Clinical, haematological, erythrokinetic and enzymatic characteristics as well as the effect of splenectomy are discussed. These studies appear to define the fundamental features of a particular disorder or a variety of hereditary elliptocytosis; characterized by a genetic autosomal dominant character, moderate degree of RBC eccentricity, erythroid dysplasia with relative marrow failure and incomplete response to splenectomy.


Effective iron chelation following oral administration of an isoniazid-pyridoxal hydrazone. When isoniazid and pyridoxal are mixed in equimolar quantities a hydrazone is formed which is able to complex with iron. The oral administration of this compound to rats in single doses of 25--100 mg/kg leads to an increase in faecal iron excretion up to 8 times the normal level. In tissue culture the compound is able to remove iron from Chang cells. The results suggest that this compound may be of potential value for the oral therapy of iron overload.


Factors influencing the treatment of patients with stage Ia carcinoma of the cervix. A total of 122 patients with Stage Ia carcinoma of the cervix was followed for 5 to 25 years. Eight recurrences were found, four of which were located in the vagina, cervix or the bladder and four on the pelvic wall. Five of the recurrences were detected after 5 years of follow-up. Tumour cells were found in blood vessels or lymphatic channels in eight patients and five of them developed metastases. It is proposed that only tumours with a depth of stromal invasion of less than 5 mm should be treated conservatively. If blood vessels or lymphatic channels contain tumour, we would recommend a Wertheim's hysterectomy or treatment by intracavitary and external irradiation.


The effects of maternally administered pethidine or epidural bupivacaine on the fetus and newborn. The continuous fetal heart rate rate pattern, condition of the baby at birth and its subsequent behaviour were compared in three groups of infants whose mothers received during labour either no drugs, intramuscular pethidine or epidural bupivacaine. The blood levels of pethidine and bupivacaine were measured at delivery in a maternal vein, and umbilical artery and vein, and in the newborn during the first 48 hours of life. The only significant changes in the fetal heart rate pattern occurred in association with maternal hypotension or uterine hyperstimulation. The Apgar scores at one minutes were 7 or less in more infants in the intramuscular pethidine and epidural bupivacaine groups as compared with the controls. There were no differences in the Apgar scores at five minutes. Neonatal behaviour during the first six weeks of life was not significantly affected by pethidine or epidural bupivacaine when compared with the control group. Pethidine and bupivacaine were shown to cross the placenta freely. The half-life of these drugs in the newborn was longer than in the adult.


Fetal breathing movements and fetal distress. Twenty-seven pregnancies were monitored by antenatal cardiotocographs, daily fetal movement counts and an assessment of fetal breathing activity by real time scanning, and the results of these tests were related to the development of fetal distress during the first stage of labour. The proportion of time during which fetal breathing movements were present, determined over only a short period of time, was found to be a useful predictor of fetal behaviour during a labour.


Fetal breathing and generalized fetal movements in normal antenatal patients. Fetal breathing movements (FBM) and generalized fetal movements (GFM) were recorded for 30 minutes using a real time B-scan instrument in 46 women with uncomplicated pregnancies. FBM occurred for 10.9+/-0.9 (SE) per cent of the recording time. The time occupied by FBM was not significantly related to smoking, meal times or plasma glucose concentrations. FBM were absent during the observation period in 15 per cent of patients and present for less than 10 per cent of the time in 33 per cent of patients.


Ultrasound assessment of fetal growth.

Ultrasound assessment of fetal growth. Consecutive ultrasonic measurements of the fetal head area, thorax area, abdomen area, head/thorax (H/T) area ratio and head/abdomen (H/A) area ratio were obtained from 100 patients with a normal pregnancy and from 186 patients with suspected intrauterine fetal growth retardation. Of all the measurements used, H/A area ratio was found to be most accurate in identifying intrauterine fetal growth retardation predicting 82.9 per cent at 33 weeks and 85.7 per cent at 36 to 38 weeks of gestation. This technique was also found to be useful in distinguishing between the two types of growth retardation. The H/A area ratio was helpful in identifying the growth acceleration pattern of a large baby.


Blood pressure changes when uterine stimulants are used after normal delivery. Use of an automatic sphygmomanometer showed that intravenous ergometrine given immediately after delivery caused significant elevation of diastolic blood pressure in all 10 normotensive women studied. Blood pressure was also raised in some patients following the use of intramuscular oxytocin and ergometrine (Syntometrine). Uterine stimulants for use after delivery must be chosen with care.


Placental protein 5 in fetal and maternal compartments. Placental protein 5 is produced by the syncytiotrophoblast and secreted into the maternal peripheral circulation reaching levels of approximately 30 micrograms per litre in normal pregnancy at term. In the present study the distribution of PP5 was examined in maternal and fetal compartments in 13 patients at delivery.


Serum gonadotrophins, prolactin and ovarian steroids in primary dysmenorrhoea. Serum samples were collected from 27 women with incapacitating primary dysmenorrhoea and from 16 normal women in the early part of the cycle (between day 3 to 6), at midcycle (between days 13 to 16) and in the late part of the cycle (between days 23 to 26) for determination of FSH, LH, prolactin (PRL), 17 beta-oestradiol (E2) and progesterone (P). The gonadotrophin and P levels showed normal and similar ovulatory patterns in both groups. The PRL concentrations (mean +/- SD) in dysmenorrhoeic women were lower than normal in the early part (9.2 +/- 4.0 ng/ml vs 14.5 +/- 7.3 ng/ml, p less than 0.01) and in the late part of the cycle (11.7 +/- 5.2 ng/ml vs 16.5 +/- 10.2 ng/ml, p less than 0.05), but not so at midcycle (9.1 +/- 2.8 ng/ml vs 10.4 +/- 4.4 ng/ml, p greater than 0.05). The E2 level was higher than normal in dysmenorrhoeic women in the late cycle (163.0 +/- 76.7 pg/ml vs 93.3 +/- 64.3 PG/ML, p less than 0.01), but apart from this the ratio of E2/P did not differ between the groups. These hormonal changes may be related to an excessive production of endometrial prostaglandins in primary dysmenorrhoea.


Immediate postpartum insertion of a copper-wired intrauterine contraceptive device, the MLCu250. The MLCu250, a copper-wired intrauterine contraceptive device, was inserted in 600 women immediately after delivery of the placenta. Thirty months after insertion, 9843 woman months of experience were computerized and the data analysed according to the life-table method. The gross cumulative pregnancy and expulsion rates were 1.3 and 6.0 respectively. No serious complications were observed and the puerperium was uncomplicated.


The effect of laparoscopic sterilisation by diathermy or silastic bands on post-operative pain, menstrual symptoms and sexuality. A total of 530 patients was reviewed six months after laparoscopic sterilisation by diathermy (235 patients) or silastic bands (295 patients). In the immediate postoperative period severe lower abdominal pain was more common in the patients who had silastic bands. There was no differences in the nature or incidence of menstrual problems when the two methods were compared. In all, 40 per cent of patients reported an increase in menstrual blood loss ad 26 per cent of patients an increase in menstrual pain, and this could not be attributed entirely to stopping oral contraception; 42 per cent of patients reported improvement in their sex lives after sterilisation, while 6.6 per cent reported deterioration mainly due to lack of libido. There was some regret about the operation in 5.1 per cent of patients.


Pregnancies in women with hyperprolactinaemia: clinical course and obstetric complications of 41 pregnancies in 27 women. Observations are reported on 41 pregnancies in 27 patients who initially had infertility and raised serum prolactin concentrations. Associated symptoms were secondary amenorrhoea (81 per cent) and galactorrhoea (81 per cent). All patients were at risk of pituitary expansion during pregnancy, especially these 19 (70 per cent) with radiological evidence of pituitary tumours. Fifteen patients had 21 pregnancies after pituitary implantation with 90yttrium; 14 patients had 20 pregnancies without prior pituitary implantation or any other attempt to prevent tumour expansion. During pregnancies, tumour enlargement as shown by diminished visual acuity, visual field defects, severe headaches, diabetes insipidus and radiological changes occurred only in 3 of the 14 patients who had not had implants. Two patients who became pregnant both before and after pituitary implantation suffered tumour expansion in their pregnancies before implantation, but not when pregnant after the operation. The induction and Cesarean section rates were about 30 per cent in 32 term pregnancies in 25 patients. Details of how pregnancy was achieved and the associated obstetric problems are given.


Enterohepatic circulation of oestriol: a study of the effects of ampicillin on plasma oestriol levels. Plasma total oestriol was measured daily before and during ampicillin therapy in five pregnancy patients and in eight patients, four of whom were receiving ampicillin, for four days following delivery. Ampicillin caused a fall in plasma oestriol of 25 per cent during the first three days of treatment, thereafter the level remained stable with a lower day-to-day variability than in untreated patients. Ampicillin reduced the plasma half life of total oestriol by 60 per cent. This study illustrates how the re-entry of oestriol from the gut leads to irregular fluctuations in plasma oestriol levels and helps to keep up the plasma concentration of the steroid when inflow from the fetoplacental unit is cut off.


The effect of maternal posture, meals and time of day on fetal movements. During the last 3 to 4 months of their pregnancies, six healthy mothers provided a daily record of their own activity together with the amount of fetal movement. Fetal movement increased throughout most of the daytime to reach a peak in the evening. The mothers detected most movement when they were lying, less when they were sitting and least when they were standing. Later in the day the mothers were less likely to be standing and more likely to be sitting; when fetal movement was measured with the mother in the sitting posture, more movement occurred as the day progressed. Mealtimes did not have any effect upon movement. Some implications of these findings, both for the assessment of fetal well-being and for theories of the development of circadian rhythms in the infant, are discussed.


Fetal movements in small-for-dates babies. Subjective assessment of daily fetal movement rates is a simple clinical guide to fetal well-being. Fetal death is unlikely to occur until rate falls below 10 movements/12 hours whilst a rate of less than 21 movements/12 hours is strongly associated with a small-to-dates fetus and indicates the necessity for further assessment of placental function.


Abortion associated with intrauterine infection by candida albicans. Case report. A 27-year-old patient who became pregnant whilst fitted with an intrauterine contraceptive device developed an intrauterine Candida albicans infection and aborted. The diagnostic (and other inconspicuous) features of C. Albicans infection in the fetus are described. The presence of the intrauterine contraceptive device may have predisposed to the establishment of the infection.


Recurrent bowel obstruction during pregnancy after ileo-jejunal by-pass. Case report. A patient weighing 140 kg had a jejuno-ileal by-pass procedure. In two subsequent pregnancies she developed bowel obstruction on a total of three occasions.


Retroperitoneal sarcomas obstructing delivery: two case reports. Two patients with abnormal fetal lies underwent elective Caesarean section. Both abnormal lies were found to be due to retroperitoneal sarcomas overlying the bifurcation of the iliac vessels. One proved to be an inoperable and radioresistant fibrosarcoma. At seven and a half years after Caesarean section and following chemotherapy this patient is alive and well and has had two more children. The other tumour was a large myxoid liposarcoma which was completely removed at the time of Caesarean section. This patient is alive and well 18 months after operation.


A compound representing the D-glycerate terminus of the methylglucose-containing polysaccharide of Mycobacterium smegmatis. In order to study the structure of the methylglucose-containing polysaccharide (MGP) of Mycobacterium smegmatis by NMR spectroscopy, we have prepared the model compound O-alpha-D-glucopyranosyl-(1 leads to 2)-D-glyceric acid. This compound, which represents the aglycon-containing terminus of MGP, was made from leucorse [O-alpha-D-glucopyranosyl-(1 leads to 5)-D-fructopyranose] by successive treatment with sodium borohydride, lead tetraacetate, and hypobromite. The structure of O-alpha-D-glucopyranosy.-(1 leads to 2)-D-glyceric acid was confirmed by chemical and enzymic methods. 13C and 1H NMR spectra of this compound, together with spectra of several disaccharides, were obtained for future reference in the polysaccharide study. The nine resonances in the 13C spectrum were assigned by comparison with the spectrum of methyl alpha-D-glucopyranoside. Analysis of the 1H NMR spectrum showed that the two methylene protons on C-3 of the glycerate moiety were less equivalent in the sodium salt than in the acid. This may be attributable to hydrogen bonding between the carboxylate and the hydrogen atom of the glycerate 3-hydroxyl group.


Biochemical properties of acteylcholine receptor subunits from Torpedo californica. Four polypeptide chains composing acetylcholine receptors from the electric organ of Torpedo californica were purified by preparative electrophoresis in sodium dodecyl sulfate. Their apparent mole ratio alpha/beta/gamma/delta is 2:1:1:1. These chains are not readily distinguished by amino acid or carbohydrate composition but are distinguished by apparent molecular weight and polypeptide maps. By peptide maps, no extensive homology is evident between these chains or between any of these chains and higher molecular weight chains found in receptor-enriched membrane fragments.


Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle. Polypeptide chains composing acetylcholine receptors from the electric organs of Torpedo californica and Electrophorus electricus were purified and labeled with 125I. Immunochemical studies with these labeled chains showed that receptor from Electrophorus is composed of three chains corresponding to the alpha, beta, and gamma chains of receptor from Torpedo but lacks a chain corresponding to the delta chain of Torpedo. Experiments suggest that receptor from mammalian muscle contains four groups of antigenic determinants corresponding to all four of the Torpedo chains. Binding of 125I-labeled chains was measured by quantitative immune precipitation and electrophoresis. Antisera to the following immunogens were used: denatured alpha, beta, gamma, and delta chains of Torpedo receptor, native receptor from Torpedo and Electrophorus electric organs and from rat and fetal calf muscle, and human muscle receptor (from autoantisera of patients with myasthenia gravis). The four chains of Torpedo receptor were immunologically distinct from one another and from higher molecular weight chains found in electric organ membranes. Antibodies to these chains reacted very efficiently with native Torpedo receptor, but the reverse was not true. Antibodies to native receptor from Torpedo and Electrophorus reacted slightly with each of the chains of the corresponding receptor. However, cross-reaction between chains and antibodies to any native receptor was most obviuos with the alpha chain of Torpedo or the corresponding alpha' chain of Electrophorus. Antiserum to alpha chains exhibited higher titer aginst receptor from denervated rat muscle. Antibodies from myasthenia gravis patients did not cross-react detectably with 125I-labeled chains from electric organ receptors. Most interspecies cross-reaction occurred at conformationally dependent determinants whose subunit localization could not be determined by reaction with the denatured chains.


Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer. The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.


Purification and the histones of Dictyostelium discoideum chromatin. Dictyostelium chromatin has been purified from nuclei in high yield by differential centrifugation and nuclease cleaving. Its chemical composition has been assayed, and its histones have been analyzed by gel electrophoresis, peptide fingerprints, amino acid composition, and ion-exchange chromatography. The mass ratios of DNA/RNA/histone/nonhistone are 1.0:0.18:0.98:1.02. There are four histones including one unusual histone, H7, which is the most abundant histone in the slime mold. The H4-like protein is the most conserved protein, while the other histones show both similarities and differences with mammalian histones.


Nuclear magnetic resonance conformational studies on the chemotactic tripeptide formyl-L-methionyl-L-leucyl-L-phenylalanine. A small beta sheet. Previous work by several groups has shown that the combination of spin--spin coupling constants and spectral density components (derived from spin--lattice relaxation and/or nuclear Overhauser measurements) may aid in the task of conformational determination of peptides in solution. Using the peptide formyl-L-methionyl-L-leucyl-L-phenylalanine, which is a potent specific chemotactic agent for leucocytes, we show the following: (a) that 3JNHCH coupling constants are consistent with a high degree of rigidity in the peptide backbone in solution, (b) that 3H isotopic substitution in combination with relaxation data taken at different Larmor frequencies enables spectral density, and thence conformational, information to be obtained, (c) that side-chain conformations for this molecule mirror, in some aspects, those found in the solid state for other peptides containing the same residues, and (d) that temperature dependence of amide chemical shifts does not have direct implication concerning the existence  of intramolecular hydrogen bonds in peptides. We are able to propose a family of conformations which appear to interchange rapidly on the NMR time scale and are characterized by a distribution of side-chain rotamers. The basic backbone conformation is, or closely approximates, a small beta antiparallel pleated sheet and as such suggests a possible mode of receptor--chemotactic peptide interaction.


Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization. Lipoamide  dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.


Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.


Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by alpha-hydroxybutynoate. The acetylenic alpha-hydroxy acid 2-hydroxy-3-butynoate (alpha HB) is a substrate and an irreversible inactivator of the FAD-containing flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. On the average, the enzyme undergoes five catalytic turnovers with alpha HB in air at pH 7.0 before being inactivated. Irreversible inactivation is due to the conversion of the flavin to a pink adduct with visible absorption peaks at 522, 382, and 330 nm and weak fluorescence with an emission maximum at 635 nm. The adduct is stable and can be released from the enzyme and purified. It retains a structure analogous to FAD since it binds to the FAD-specific apo-D-amino acid oxidase. It can be further converted to an FMN analogue with phosphodiesterase which binds to the FMN-specific apoflavodoxin. Experiments were conducted to test whether inactivation was initiated by an alpha HB allene carbanion or the dehydrogenation product of alpha HB. Kinetic studies proved inconclusive in that a rapid equilibrium between an oxidized enzyme--allene carbanion pair and reduced enzyme--keto acid pair would make these two species kinetically equivalent. The olefinic substrate 2-hydroxy-3-butenoate, however, produced no flavin adduct. Since the keto acid derived from the oxidation of this alpha-hydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the alpha-hydrogen of alpha HB is the reactive species which covalently adds to the flavin.


Structure of the flavin adduct formed in the suicide reaction of alpha-hydroxybutynoate with D-lactate dehydrogenase. The Zn-dependent flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii is irreversibly inactivated by the D form of the suicide substrate 2-hydroxy-3-butynoic acid. The process of inactivation involves formation of a new pink chromophore, which can be released in intact form from the protein and which was purified to homogeneity by affinity chromatography. Inactivation involves covalent addition of the suicide substrate to the flavin coenzyme. The optical spectra indicate an elongation of the flavin chromophore, and the chemical reactivity suggests a derivative of reduced flavin. The structure of this adduct was deduced from Fourier transform NMR, from the chemical properties, and from comparison with appropriate models, which were synthesized chemically. This structure involves the covalent linkage of the acetylenic inhibitor to positions N(5) and C(6) of the flavin coenzyme via carbon atoms 2 and 4 of the inhibitor to form an additional fused aromatic ring. The pink adduct can be reconverted to an isoalloxazine chromophore by reduction with borohydride and subsequent reoxidation with oxygen. This new isoalloxazine has the spectral properties of an isoflavin, and it is proposed to carry the moiety of the inactivator molecule as substituent at position C(6). The structure of the pink chromophore representing a cyclic adduct to the flavin positions N(5) and C(6) is compared to that of the adduct obtained from L-lactate oxidase from Mycobacterium smegmatis and the L form of the same inhibitor [C(4a)--N(5) cyclic adduct; Schonbrunn, A., Abeles, R. H., Walsh, C. T., Ghisla, S., Ogata, H., and Massey, V. (1976) Biochemistry 15, 1978]. This comparison allows deductions about the relative orientation of substrate, coenzyme, and active center functional groups in the two enzymes.


Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale. We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.


Physical properties of collagen--sodium dodecyl sulfate complexes. Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.


Effects of internal nonbonded bases and a G.U base pair on the stability of a short ribonucleic acid helix. Proton nuclear magnetic resonance has been used to examine the effect of both noncomplementary and G.U oppositions in the duplexes formed by the synthetic pentaribonucleotides CpApApUpG, CpApUpUpG, CpApGpUpG, and CpApCpUpG. The lack of any sigmoidal behavior in the chemical shift vs. temperature plots of the base protons in the individual pentaribonucleotides indicates that duplexes with noncomplementary base oppositions of the type: formula: (see text), (where X = A, U, G, or C) do not form. Variable temperature spectra of the mixture of CpApGpUpG and CpApUpUpG were recorded over the range of 70--10 degrees C. The chemical shift vs. temperature plot of the purine aromatic protons displayed sigmoidal curves. This demonstrated both duplex formation and the presence of a G.U. base pair. The average Tm of the duplex was found to be 23.4 +/- 2.0 degrees C. This is similar to that of the duplex formed by CpApUpG (24.0 +/- 1.0 degrees C) but less than the Tm of the following duplexes: CpApApUpG:CpApUpUpG (Tm = 28.5 +/- 2.1 degrees C), CpApGpUpG:CpApCpUpG (Tm = 38.4 +/- 0.6 degrees C) and CpApUpApUpG (Tm = 41.5 +/- 1.1 degrees C). The G.U base pair has a Tm (20.0 degrees C) significantly lower than the rest of the duplex (24 +/- 1 degree C) and is a region of local instability within the double helix. This 1H NMR study is the first to investigate both the formation and relative stability of an internal G.U. base pair neighboring regular Watson--Crick base pairs.


Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates. A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.


Primary structure of rabbit alpha-lactalbumin. Rabbit alpha-lactalbumin was purified from the milk of New Zealand White rabbits. It was found to exist predominantly as a glycoprotein, containing 5 mol of glucosamine per mol of protein, as well as other sugars. The amino acid sequence of the protein was determined by sequenator analysis and carboxypeptidase digestion. There are 122 amino acids in the protein and a single carbohydrate moiety, probably attached to an asparagine residue at position 45. The C terminus of rabbit alpha-lactalbumin is one residue shorter than that of the other alpha-lactalbumins. Sequence comparisons indicate that the alpha-lactalbumin gene has been undergoing more frequent mutation than had previously been thought. A new method of preparative peptide mapping using 2,5-diphenyloxazole (PPO) fluor to detect peptides is described.


Increased thermal stability of proteins in the presence of sugars and polyols. Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.


Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide. The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...


Correlative statistical analysis and computer modelling of intramembraneous particle distributions in human erythrocyte membranes. The planar distribution of intramembranous particles on the P faces of freeze-fractured human erythrocyte membranes is characterized by radial distribution, angular distribution and differential density distribution analysis. Various degrees of intramembranous particle aggregation induced by spectrin removal and low pH are differentiated through computation. Random hard disk models with various disk diameters are built for comparison studies. In all samples, the 80 +/- 10 A particles are found to have a preferred neighboring distance of 100 +/- 10 A, but no preferred angular relation is found between neighboring particles. A pattern recognition process using both radial and density distribution analyses reveals that none of the particle distributions observed may be regarded as random. The fact that the particle distributions observed are neither even nor random suggests that factors other than long range electrostatic force alone are involved in determining the particle distribution.


Dielectric noise in membranes.

Dielectric noise in membranes. Most theoretical and experimental studies of electrical fluctuations in membranes so far have been devoted to noise associated with conduction processes. In this paper a different type of noise is described which results from dipolar transitions in the membrane. Two mechanisms for the generation of such dielectric noise are analyzed: (a) conformational transitions of membrane proteins involving changes in dipolar moment and/or polarizibility, and (b) rotation of dipolar molecules dissolved in the lipid. The spectral intensity of current noise calculated for the two models exhibits a characteristic dependence on frequency omega with a decrease proportional to omega 2 towards low frequencies and an approach to a frequency-independent (white noise) limit at high frequencies. For a given number of dipolar molecules in the membrane, the spectral intensity is inversely proportional to the square of the membrane thickness.


Planar bilayer membranes from pure lipids. Planar lipid bilayer membranes are formed from mixtures of pure lipids in the absence of non-biological solvents. The solventless bilayers are characterized by a large specific capacitance (586-957 nF/cm2) comparable to that of cell membranes but considerably greater than that of conventional lipid/decane bilayers. Hydrocarbon solvents, such as n-alkanes or squalene, thicken the bilayer. Membrane dielectric thickness is used as an indicator of bilayer lipid composition. For membranes made from pure monoglyceride/triglyceride mixtures the thickness of the solventless lipid bilayer is independent of both the chain length (11-22 carbons) and mol fraction (0.1-0.9) of triglyceride in the bulk mixture. In contrast, the thickness of the bilayer (2.0-3.3 nm) depends strongly upon the length (16-24 carbons) of the monoglyceride component. Molecular volume considerations lead to the conclusion that the bulk lipid mixture disproportionates to yield bilayer membranes composed of nearly pure monoglyceride. The dielectric thickness of the monoglyceride bilayer is consistent with the notion that the lipid fatty acyl chains are fluid.


Block poly(Ala)-poly(Lys). A water-soluble model for intrinsic membrane proteins? Block poly(Ala)16-poly(Lys)13.5 was synthesized by the Leuchs anhydride method. This polypeptide is water soluble in a largely monomeric form, but binds rapidly and spontaneously to unilamellar vesicles of dimyristoyl phosphatidylcholine at pH 7.4. The interaction is evidently of a hydrophobic nature since the complex is not disrupted by salt and no similar reaction is given by polylysine. Evidence for the interaction was obtained by ultrafiltration, chromatography on Sepharose 4B, and sedimentation velocity ultracentrifugation. While direct information on the molecular structure of the complex is still lacking, we propose that this amphipathic block copolymer binds to lipids in a similar manner as intrinsic membrane proteins and hence can be used to study the interactions of intrinsic proteins with lipids.


Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles. Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca2+ and K+ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163-202). The Ca2+-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and bindng to negatively-charged groups on the membrane surface. The K+-induced reaction includes only dimerization and is shown to be due to screening interactions alone. The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca2+-induced reaction has a 'bimolecular' rate constant of 4.77 . 10(8) M-1 . s-1. These values are close to the limit of diffusion control (8.03 . 10(9) M-1 . s-1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca2+-induced aggregation showed a break at 5 degrees C. Above this temperature, the activation energy is low (+0.65 kcal/mol), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing theenergetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of 'recognition sites' on the membrane surface. The results were analyzed in terms of an encounter complex model in which vesicles with separations of 26-126 A are considered capable of transformation into a stable complex. The rate constant of the transformation step is 1.4 . 10(3) s-1 for Ca2+ and approx. 1.6 . 10(5) s-1 for K+. The values are compared with previous results for chromaffin granule membranes and for phospholipid vesicles derived from chromaffin granule lipids and from acidic phospholipids. The half-time for Ca2+-induced transformation of the encounter complex into the stable complex is 435 microseconds. It is concluded that the recognition sites are almost as optimally deployed as the vesicle plasma membrane recognition sites involved in exocytotic release.


Effect of anions on folate binding by isolated brush border membranes from rat kidney. The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3',5',9-3H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70-75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3',5',9-3H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroyl-glutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroyl-glutamic acid. Inhibition of [3',5',9-3H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3',5',9-3H]pteroylglutamic acid.


Vitamin A deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes. Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5-10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R1-6 has an absorbance maximum at about 490 nm, but no unusually enhanced beta-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.


Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid. The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.


Cation-induced asymmetry of choline flux across presynaptic plasma membranes. Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K+ and extrude Na+ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na+, Li+, Rb+, Cs+ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na+ gradient gave a maximal value of about 10(2). A secondary active transport of choline, where Na+ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.


Developmental and other characteristics of lysine uptake by rat brain synaptosomes. Synaptosomes isolated from adult or newborn rat cerebrum take up L-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is mort tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher Vmax than those of the adult for high affinity system but adult for high affinity system but adult synaptosomes have a higher Vmax than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low Km system for lysine uptake.


The effect of diamide on amino acid transport by rat renal cortex slices. Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.


The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes. The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitiors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces , is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.


Plasma membrane aminophospholipid distribution in transformed murine fibroblasts. The possibility that the asymmetric distribution of aminophospholipids may be an intrinsic property of mammalian plasma membranes was examined in LM cells, a transformed murine fibroblast cell line. The cells were grown in suspension culture in a chemically defined medium without lipid, protein, or serum and then treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). A maximum of 4% of LM cell plasma membrane phosphatidylethanolamine and 5% of the phosphatidylserine was labelled with TNBS. Furthermore, long chain and unsaturated fatty acids were preferentially esterified to the non-derivatized phosphatidylethanolamine (inner monolayer) as compared to phosphatidylethanolamine derivatized with TNBS (outer monolayer). Isethionyl acetimidate, an alternative non-penetrating reagent, confirmed the results obtained with TNBS and provided supportive evidence for the highly asymmetric distribution of phosphatidylethanolamine; 6% of the phosphatidylethanolamine was labelled with isethionyl acetimidate. When the penetrating reagent methylacetimidate was used, more than 80% of the phosphatidylethanolamine was derivatized. Although the growing of the LM cells in 10% calf serum significantly increased plasma membrane phosphatidylcholine while decreasing phosphatidylethanolamine, calf serum had no significant effect on phosphatidylethanolamine or phosphatidylserine asymmetry.


In vitro interaction of Zajdela ascites hepatoma cells with lipid vesicles. We studied the in vitro interaction between Zajdela ascites hepatoma cells and small unilamellar vesicles, consisting of 14C-labeled phosphatidylacholine, cholesterol, and phosphatidylserine (molar ratio 5 : 4 : 1), containing high intravesicular concentrations of carboxyfluorescein or fluorescein isothiocyanate tagged dextran. The entrapped markers were found to be associated with the cells to a lesser degree than the vesicle membrane marker. This discrepancy, which is slightly less pronounced for fluorescein isothiocyanate tagged dextran than for carboxyfluorescein, increases with incubation time and decreases with increasing vesicle lipid concentration in the incubation mixture. Vesicle-plasma membrane exchange of the vesicle lipid marker could not entirely explain the observed discrepancy. It is tentatively concluded that the gap mainly arises from a selective loss of entrapped dyes from vesicles actually interacting with the cell surface. Both spectrofluorimetry and fluorescence microscopic observations, as well as the relative insensitivity of vesicle uptake towards the presence of metabolic inhibitors, exclude a major contribution of endocytosis as a vesicle uptake route. We therefore conclude that vesicles are primarily internalized by a vesicle-cell fusion-like process. The observed discrepancy in uptake between entrapped materials and vesicle lipid is discussed in terms of a two-site vesicle-cell surface interaction model.


The influence of cholesterol on head group mobility in phospholipid membranes. The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0--70 degrees C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42 degrees C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42 degrees C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42 degrees C, however, a decrease in f2 at cholesterol contents up to 20--30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.


The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium. The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.