Breast cancer rates in populations of single women.

The well known associations of breast cancer with fertility patterns and diet are interdependent and it is difficult to estimate the extent to which breast cancer is related to diet. This was attempted by analysing breast cancer rates in populations of single (never married) women for which the contribution of childbearing would be small. Age specific breast cancer rates for single women showed the same variation by country, social class, urban-rural area and with time, as did the corresponding rates for married women, suggesting that common or related factors determined breast cancer rates in single and married women. Also, dietary correlations of breast cancer rates at 55-64 years, around 1960, were not sifnificantly different for single women and the general female population. This supported the view that the dietary associations with breast cancer, observed in larger studies of general female populations, did not arise indirectly from an association with childbearing rates. It was pointed our that the positive association of breast cancer with sugar, observed for single and for all women, was accopanied by a negative association with starch. These opposite associations with two forms of varbohydrate seemed inconsistent on general nutritional grounds and could be explained as arising indirectly to the association of breast cancer with affluence. Otherwise, it would seem necessary to establish a nutritional difference between starch and sugar, which could reasonably influence breast cancer rates, before the association was accepted as indicating cause.


Further analysis of the anti-tumour effect in vitro of peritoneal exudate cells from mice treated with Corynebacterium parvum.

Administration of C. parvum to both intact and thymectomized mice resulted in the appearance in the peritoneal exudate of cells which inhibited tumour growth in vitro. This effect was mediated by intact, viable adherent cells, which it seems reasonable to categorize as macrophages, and was contingent on contact between the effector and target cells. No co-operation was observed between lymph node cells from C. parvum treated mice and peritoneal exudate cells from normal mice.


Quantitative studies of translymphnodal passage of tumour cells naturally disseminated from a non immunogenic murine squamous carcinoma.

A squamous cell carcinoma of spontaneous orgin in a WHT/Ht mouse was used to study the frequency with which the regional axillary lymph nodes draining subcutaneous or intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. This frequency (similar to 40%) was found not to vary significantly with the size or duration of the tumour drained and not to be increased by coincident infective, traumatic or antigenic stimuli acting at the tumour site or in adjacent tissue. Because tumour growth occurred in only 2/55 (4%) nodes which were left in situ in mice whose tumours were radically excised, it was concluded that tumour forming node transplants reflected a small and limited content (estimated to be about 13) of transnodally passing tumour cells destined to pass on to the blood; separate experiments showed that tumour cells reaching the blood survived for only a few hours. Nodes from tumour-excised mice gave rise to tumours as frequently when autografted as when isografted to mice with no previous expose to the tumour. A review of the finding reported here and of previous quantitative data for this system enabled us to exclude any implication of anti-tumour immunity from our interpretation of the results of the experiments.


The growth kinetics of xenografts of human colorectal tumours in immune deprived mice.

The technique of labelled mitoses was used to examine cell proliferation within grafts of human colonic and rectal tumours in immune deprived mice. Most of the data were obtained on the first passage but in some cases up to the third passage was used. It was found to be difficult to obtain precise kinetic data on this type of tumour material, but the results did allow some estimates to be made, particularly of the duration of the G2 and S phases of the mitotic cycle. The average G2 duration was 6 h and the average S phase was 14 h. It is concluded that whilst xenografts may differ in a number of respects from the tumour in the patient, they nevertheless constitute a type of experimental tumour that is worthy of further study.


Venous diversion trapping and growth of blood-borne cancer cells en route to the lungs.

A proportion of W-256 tumour cells injected intravenously into a tail vein of the rat are diverted into venous plexuses en route to the lungs; here tumour cells remain trapped, proliferate and form invasive solid tumours in the pelvis and hindquarters, which cause paraplegia, metastases and death. Also, cells trapped in veins produce tumour nodules distributed along the length of the tail; this effect in markedly enhanced by temporarily arresting the outflow of blood from the tail for a few seconds only immediately after cells are injected. Continous monitoring of the radioactive signal over the lungs after W-256 cells labelled with 125IUDR were injected showed that massaging the tail or intravenously injecting isotonic saline into the tail dislodged cells trapped in veins. In heparinized rats, tail trapping was markedly reduced, although not entirely abolished, and venous trapping in vertebral and pravertebral regions was decreased. The anatomical distribution of growth of the trapped cells in rats closely resembled metastases involving dissemination via the "vertebral venous system" produced by certain cancers in man. Labelled tumour cells trapped in the lungs of untreated mature rats commenced dying rapidly in situ wiht 1-2 h after injection; the majority had disappeared within 24 h, and less than 1% of the injected tumour cells survived to form lung colonies. Experimental evidence is presented which indicates that the lungs play a vital role in rapidly eliminating a high proportion of blood-borne cancer cells in the adult individual.


Influence of ICRF 159 and trition WR 1339 on metastases of a rat epithelioma.

ICRF 159 and Triton WR 1339 have been examined for their ability to suppress subcutaneous growth and pulmonary metastases from a transplanted rat epithelioma. Neither compound influenced subcutaneous tumour development or reduced the propensity to metastasize when administered in regimens reported to suppress pulmonary, lymph node or intracerebral metastases in other experimental system.


The ultrastructure of lymphoblastoid cell lines from Marek's disease lymphomata.

The ultrastructure of two lymphoblastoid cell lines derived from Marek's disease lymphomata has been studied. The cells varied from 5 to 12 mum in diameter and had large round or oval nuclei. A nucleolus was occasionally present and about 3% of cells showed projections of the nuclear envelope. The cytoplasm contained many ribosomes and several mitochondria but endoplasmic reticulum was sparse. A small number of cells contained annulate lamellae and crystalline structures were occasionally seen. Cells with immature intranuclear herpesvirus particles were rarely present. The cells had many ultrastructural features in common with Burkitt's lymphoma-derived cell lines.


Human studies with "high dose" metronidazole: a non-toxic radiosensitizer of hypoxic cells.

The serum concentrations of the radiosensitizer metronidazole have been determined in mice for both oral and intraperitoneal doses of the drug and these have been related to radiosensitization studies in murine tumour systems. In preliminary work before a possible clinical trail the serum metronidazole concentration/time curves have been determined in 7 patients using single doses of metronidazole of up to 15 g. The data suggest that a linear relationship exists between the metronidazole dose expressed in mg/kg and the peak serum concentration. The possibility of achieving radiosensitization of tumours in patients after tolerable doses of metronidazole is discussed in relation to enhancement ratios determined for in vitro and in vivo systems. It is concluded that predictions from in vitro systems give values that are probably too optimistic.


Modest radiosensitization of solid tumours in C3H mice by the hypoxic cell radiosensitizer NDPP.

The x-ray dose required to cure half the mice bearing first generation transplanted mammary carcinomata 150 days after irradiation was determined. NDPP proved to be a relatively poor radiosensitizer in mice, for although a maximum enhancement ratio of 1-3 was obtained when x-rays produced from a 1-4 MeVp electron accelerator were given between 10 and 17 min after the administration of NDPP, this was at a drug concentration sufficient to cause marked kidney abnormalities in 5-10% of the mice.


Assay of anti-cancer drugs in tissue culture: conditions affecting their ability to incorporate 3H-leucine after drug treatment.

An attempt has been made to construct an assay potentially suitable for use with primary cultures of human tumours to measure the survival of exponentially growing monolayer cultures after exposure to anti-neoplastic drugs. Cell survival was assessed using their protein synthetic capacity after removal of drugs. HeLa cells were employed to avoid the ingerent variability and heterogeneity of primary cultures from human tumours, and an assay has been constructed using microtitration trays to provide large numbers of replicate cultures without the requirement of a large number cells. An increase in the duration of the exposure to drug increased sensitivity in nearly all cases examined. Similarly, an increase in the period of culture following drug removal produced increased sensitivity to alkylating agents but allowed recovery from exposure to certain cycle-dependent drugs. Some of the drugs used were shown to be unstable under culture conditions and vinblastine was actively metabolized, although this instability was not necessarily reflected in the time course of the drug's effect. Mustine sensitivity was shown to be reduced by an increase in cell density at a level where density limitation of 3H-thymidine incorporation becomes apparent. These variations and possible methods of minimizing their effects are discussed.


Pre-natal irradiation and childhood malignancy: a review of British data from the Oxford Survey.

This paper reviews data relating to obstetric radiography from the Oxford Survey of Childhood Cancers, i.e. for deaths in Britain from 1953 to 1967. Some 8513 cases were traced and used in the analyses, together with an equal number of matched controls. The relative risk estimate (1-47 overall) does not vary significantly between different tumour groups, for different ages at death, nor between sexes. Other epidemiological factors-sibship position, maternal age, social class, region of residence and maternal morbidity-are analysed and show varying degrees of association, but not sufficient to "explain" the observed risk in terms of a selection effect. The dependence of the risk on the number of films exposed is highly significant and adequately described by a linear relationship. The timing of and reason for the exposure are also examined. Analysis of the risk by year of birth shows a pattern of steadily declining risk for both solid and haematopoietic tumours; this may be partly attributable to lower radiation doses per film exposed but is also due to the smaller numbers of films used. A consequence may well be that the risk-always of small clinical significance-would become virtually undetectable in future investigations.


Enhancement of syngeneic murine tumour transplantability by whole body irradiation--a non-immunological phenomenon.

Experiments were undertaken to test the general validity of the assumption that potentiation of tumour transplantability by sublethal whole body irradiation (WBI) implies some degree of immunological resistance in the intact host. A transplantable carcinoma of spontaneous origin in CBA mice which exhibits a large WBI effect was assayed quantitatively in mice which had been immunologically crippled in terms of allograft acceptance by depletion of thymus derived lymphocytes. The mean number of tumour cells required for 50% successful takes (TD50) in these mice was found to be not significantly different from that in normal controls but highly significantly greater than in WBI mice. On the other hand, in mice which underwent laparotomy immediately before assay, the TD50 was reduced significantly though not to the same extent as in WBI mice. It was concluded that WBI effect was not due to impaired host immunity but possibly to physiological changes resulting from acute stress. The hypothesis that hyperfibrinogenaemia which occurs after both WBI and laparotomy might increase tumour transplantability was rejected because of the lack of correlation between TD50 and fibrinogen levels at different times after each procedure. From this and other work it is apparent that TD50 data, in themselves, give no reliable indication of host immunity.


The influence of tumour burden and therapy on cellular cytotoxicity responses in patients with ocular and skin melanoma.

Using a microassay for cellular immunity, tumour specific cytotoxicity was detected in 2/5 cases of ocular melanoma and 1/3 cases of primary cutaneous melanoma before treatment. Reactivity was measured against allogeneic skin melanoma target cells in short or long term in vitro culture. Lymphoid cells from patients with disseminated cutaneous melanoma were either non-reactive (4/8 cases) or gave a nonspecific cytotoxicity on target cells of diverse histogenic origins. Among tumour-free patients tested after surgery, 0/2 patients with ocular tumour were non-reactive 3-4 months post surgery. After sugical excision of cutaneous melanoma 2/2 patients gave tumour specific reactions during the first month after surgery. After longer time intervals, from 5 months to 3 years, only 1/8 patients were reactive. Preoperative radiotherapy in a total skin dose of 10,000 rad produ-ed a transient tumour specific reaction 24 h after therapy in a single case. Following local tumour excision in patients given preoperative irradiation, 2 cases which had previously demonstrated tumour specific CMI lost reactivity. Among 14 tumour-free individuals tested only after preoperative radiotherapy and surgery, at intervals from 5 day to 13 years, a single case gave tumour specific CMI. Palliative irradiation in doses 4000-4960 rad to the inguinal or axillary lymph nodes was found to induce a generalized lymphopenia within 48 h after treatment. Lymphoid cell preparations from patients with localized melanoma contained significantly increased numbers of immature cells (lymphoblasts and myeloblasts) and myeloid precursor elements. Those prepared from patients with disseminated disease had in addition elevated levels of eosinophils but reduced numbers of recoverable lymphocytes.


An assessment of the effects of hormones on short term organ cultures of human breast carcinomata.

Twenty-eight mammary carcinomata were maintained in organ culture in the presence of various hormones. The effects of the hormones have been assessed histologically by estimation of total dehydrogenases activity of the pentose glycolytic pathway and by the incorporation of tritiated thymidine or uridine into DNA or RNA. No significant effects on tumour cell activity due to hormones have been observed.


Spontaneous and chemically induced transformation of rat embryo cell cultures.

The transformation of Wistar rat embryo cells in vitro has been studied in passaged cultures using two criteria for transformation: (1) the ability of cells to form colonies in soft agar and (2) the ability of cells to form tumours in young syngeneic animals. In general there was good correlation between the two criteria. Spontaneous transformation was observed in all untreated cultures by 44 weeks although, by not allowing the cells to become confluent, the tendency was for cultures to transform earlier (i.e. 15-21 weeks). It was noticeable that despite untreated cultures having been in vitro for different lengths of time, most cultures transformed after a similar number of passages (42-50). Treatment of the embryo cells with the alkylating agent nitrosomethylurea (NMU) or benzo(alpha)pyrene (BP) sometimes resulted in transformation after a shorter period in vitro than the controls (minimum 12 weeks) although some treated cultures took longer. Transformed cells produced transplantable fibrosarcomata in syngeneic hosts and those arising from NMU transformed cells were histologically different from those arising from spontaneously transformed cells. The significant of spontaneous transformation in in vitro rat cell transformation systems is discussed.


Temperature sensitive cells in the study of carcinogenic transformation.

Two temperature sensitive variants (ts13 and ts14) of an African green monkey tetraploid kidney cell line (epithelial), carrying temperature sensitive lesions in thymidine metabolism, were transformed by methylnitrosourea (MNU) at the permissive temperature of 33 degrees C, nor was there any transformation in the wild type parental cell line of BSC-1 at these temperatures under similar conditions. A comparative study of the cell cycle and metabolic efficiency in the 3 cell lines was performed in order to get an understanding of the physiology of the "target cells" in culture. Compared with the parental cell line of BSC-1, ts13 and ts14 cells were blocked in the G1 phase of the cell cycle during the time that the cell were in contact with the carcinogen (MNU); the variant cells also had higher mitotic indices at this time. The cells of ts13 which showed 50% more transformation than those of ts14 differed from the latter in having larger numbers of viable cells arrested in mitosis over the G1 period. The results were interpreted to indicate that there were other factors, besides cells arrested being in G1, which contributed to the difference in the frequency of transformation between the variant cell lines which had an otherwise similar physiology. Using gel electrophoresis a new protein was located in the nuclei of the transformed cells of ts13 and ts14 which was absent in the wild type cell line of BSC-1 or in the variants ts13 and ts14 at 39-5 degrees C.


Cell kinetics of urethane induced murine pulmonary adenomata: I. The growth rate.

A single injection of urethane into adult male A2G mice produced an increase in the proliferative rate of alveolar wall cells, reaching a peak at 2 weeks post urethan (PU) and declining to control levels by 2 months PU. During this urethane induced proliferative response the single and double labelling indices and the native metaphase index were all elevated although there was no corresponding alteration in the arrested metaphase index. This proliferative response may not be restricted to hyperplasia of potentially neoplastic cells, such as type II epithelium, but may also include type I epithelial cells and alveolar macrophage precursors. However, it was impossible to identify individual cell populations by methods used. The growth rate of adenomata decrease with time and cell kinetic techniques showed that the rates of entry of adenoma cells into DNA synthesis and into metaphase were decreasing concurrently with the growth rate. Thus the rate of cell production falls as adenomata age but how much cell loss contributes to the decrease in growth rate is not yet known. Decreasing cell production could be due to an increased cell cycle time and/or a decreased growth fraction. The duration of DNA synthesis in adenomata increased markedly as the mice survived, suggesting that the cell cycle time might be increased, but further experiments are required to determine whether the growth fraction changes. Attention is drawn to a complication that Colcemid introduces into kinetic studies on alveolar wall cells.


Changes in sensitivity to cytotoxic agents occurring during the life history of monolayer cultures of a mouse tumour cell line.

Dose-response curves have been obtained for the response of EMT6 mouse tumour cells in vitro to adriamycin, actinomycin D, nitrogen mustard, BCNU and CCNU. The experiments have been carried out with exponentially growing cells and with cells in early and late plateau phase. The results are discussed with particular reference to discrepancies with the results obtained by other workers in similar systems.


Biosynthesis of 17beta-oestradiol in human breast carcinoma tissue and a novel method for its characterization.

Conversion of 7alpha3H-testosterone to 17beta-oestradiol by human mammary carcinoma tissue in vitro has been demonstrated. It was characterized unequivocally by conversion to 17beta-oestradiol-3-sulphate upon incubation with adenosine-3'-phosphate-5'-phosphosulphate and the highly specific enzyme oestrogen sulphotransferase.


Response of rat lung to 3,4-benzpyrene administered by intratracheal instillation in infusine with or without carbon black.

In a controlled experiment groups of SPF Wistar rats were give 18 once-fortnightly doses of 0-5, 1-0 or 2-0 mg 3,4-benzpyrene (BP) suspended in infusine (I) or in carbon black (CB) + I by intratracheal instillation. Of rats examined post mortem, 1/16 given I+CB only, 0/16 given I only, 15/51 given BP in I+CB and 24/48 given BP in I developed squamous neoplasms of the lung. The incidence of tumours was significantly related to dose of BP. At the 1 or 2 mg dose levels BP in I only was more productive of tumours than BP in I+CB. Other changes encountered included squamous metaplasia of alveolar and bronchiolar epithelium (Sq.M), but not of bronchial epithelium, and cuboidal and columnar metaplasia of alveolar epithelium in the vicinity of terminal bronchioles (CCM). Sq.M was associated with exposure to BP or I+CB. CCM was strongly associated with exposure to I+CB but only weakly with exposure to BP.


Response of rat lung to tobacco smoke condensate or fractions derived from it administered repeatedly by intratracheal instillation.

The repeated intratracheal instillation of cigarette smoke condensate (SWS) in rats at close to maximum tolerated dose levels failed to induce squamous neoplasms in the lungs although such treatment was associated with an increased incidence of cuboidal/columnar metaplasia (CCM) and squamous metaplasia (Sq.M) of alveolar epithelium. With one exception, various fractions of SWS had no effect on lung tumour incidence though some were more effective than SWS in increasing the incidence of CCM and Sq.M. The exceptional fraction, Fraction P, which contains most of the polycyclic aromatic hydrocarbons of smoke and is the most effective of the fractions tested in producing tumours in mouse skin, gave rise to 4 squamous tumours of doubtful malignancy and one metastasizing squanmous carcinoma among 3 groups of 18 animals exposed at 3 different dose levels. The results are discussed in relation to the possible development of a method for comparing condensates for relative lung carcinogenicity.


Response of rat lung to inhaled vapour phase constituents (VP) of tobacco smoke alone or in conjunction with smoke condensate or fractions of smoke condensate given by intratracheal instillation.

In a controlled experiment, 6 groups of SPF rats were given cigarette smoke condensate (SWS) in solid form without a vehicle once fortnightly by intratracheal instillation, at 3 dose levels with or without additional exposure to the vapour phase of smoke (VP) from 10 plain cigarettes each week. Treatment continued for life. Six other groups were similarly treated with one of 3 fractions of condensate with or without VP. Exposure to VP was associated with a significant reduction in body weight, but not signficantly with the incidence or severity of any observed pathological change in the lungs. A significant dose-related assoication was seen between SWS or its fractions and the incidence and degree of chronic respiratory disease (CRD), cuboidal or columnar metaplasia (CCM) and squamous metaplasia of alveolar epithelium (Sq.M) produced. No neoplasms, however, were elicited. A significant correlation was found between the degrees of CCM and of Sq.M produced in the 24 groups exposed to SWS or fractions. The results are discussed in the light of studies in which rats were exposed to tobacco smoke by inhalation and of studies in which the same condensate and fractions were applied to mouse skin.


Response of rat lung to inhaled tobacco smoke with or without prior exposure to 3,4-benzpyrene (BP) given by intratracheal instillation.

SPF rats were exposed to the smoke from 10 cigarettes per week from the age of 10 weeks until they died. Survival, body weight, tumour incidence and histopathological appearances of the lungs were compared with those for untreated sham exposed rats. Two further groups were given a single dose of 3,4-benzyprene (BP) by intratracheal instillation. One of these was then exposed to the smoke of 10 cigarettes per week till death. Compared with untreated or sham exposed rats, exposure to smoke was associated with a significant reduction in incidence of mammary tumours. Exposure to smoke was associated with an increasing incidence of collections of macrophages laden with golden-brown pigment (GBM) and of areas of cuboidal or columnar metaplasia (CCM) or squamous metaplasia (Sq.M) of alveolar epithelium. In control rats there was virtually no GBM, a low incidence of CCM and Sq.M. Four out of 406 smoke exposed rats which came to post mortem had squamous neoplasms in the lungs, 3 having lesions of doubtful malignancy and one having a squamous carcinoma. In contrast, no squamous neoplasms were seen in 197 control rats. This difference was not statistically significant. The findings in rats given a single dose of BP were, in all the above respects, similar to those in untreated rats, except that one developed a squamous carcinoma of the lung. The effects of a single dose of BP followed by smoke exposure were in general similar to those of smoke exposure only. Three rats on this treatment regimen developed squamous cancers of the lung. None of the treatments increased the incidence of adenomata of the lungs. The results are discussed in relation to other studies of the effects of smoke exposure on rats and other species.


Intestinal polyps in American negroes and Nigerian africans.

Forty Africans from Nigeria and 89 American negroes with colorectal polyps were analysed by age, sex and type of polyp. The Nigerians were much the younger group (mostly under 20 years of age, whereas most of the American negroes were over 50) and far fewer of their polyps were truly neoplastic (7.5% compared with 87% of the Americans). These differences may be partly due to the American negro population being older than the Nigerian, and partly to environmental factors like those previously postulated to account for the difference in colon cancer incidence between these populations.


The monolayer and organ culture of human colorectal carcinomata and the associated "normal" colonic mucosa and their production of carcinoembryonic antigens.

The carcinoembryonic antigen (CEA) was produced and released by human colorectal carcinomata and also the normal attached mucosa when maintained in both monolayer and organ culture. Immunoperoxidase cytochemical methods were employed for the cellular localization of CEA which was demonstrable only on the neoplastic cells. Gel filtration and immunological methods confirmed that CEA, produced by normal and neoplastic cells, had properties similar to "authentic" CEA derived from metastatic colorectal carcinomata. In addition, two other CEA cross-reacting macromolecules, neither of which was CCEA-2, were produced by these tumours in culture.


Hepatitis Bs antigen and liver cancer: A population based study in Kenya.

Peers and Linsell (1973) demonstrated a significant association between the incidence of primary liver cancer and ingested aflatoxin in a study in the Muranga district of Kenya. A study of hepatitis B antigen in the same district showed no significant differences between the low altitude area, with a relatively high incidence of primary liver cancer, and the high altitude area with a lower incidence of the tumour. Current evidence is more in favour of aflatoxin playing an important role in the aetiology of primary liver cancer but hepatitis B antigen may play an ancillary role.


Electrical potential difference between basal cell carcinoma, benign inflammatory lesions and normal tissue.

Measurement of the resting electrical potential difference on the surface of the skin has been made on patients with suspected basal cell carcinoma. It was demonstrated that in fifty-five lesions from forty-nine patients, the mean electrical potential difference from thirty-six basal cell carcinomas was found to be +II mV, from nineteen benign inflammatory lesions it was found to be +2 mV, and from normal skin, contra-lateral to the lesions in both groups, it was found to be -3 mV. All the measurements are relative to a reference electrode placed under the tongue. The difference between the means for the inflammatory lesions and for the normal skin was found to be statistically insignificant, but the difference between these means and the mean potential difference for the basal cell carcinoma group was found to be highly significant (P less than 0-1%). However, the populations overlap to such an extent as to make the measurement, in its present form, useless as a diagnostic technique.


Coeliac disease with primary cutaneous amyloidosis.

A case is described where the patient suffered from coeliac disease, and a widespread eczema which was succeeded by lichen amyloidosus.


Atopic disease and serum immunoglobulin-E.

Fifty-seven subjects, forty-three with various combinations of atopic disease and fourteen non-atopic controls, were studied using a battery of immediate skin test allergens and a radio-immunoassay for serum Immunoglobulin-E (IgE). The geometric mean serum IgE level (units/ml) was 50 in strictly nonatopic controls, 170 in atopic respiratory disease (ARD) patients, 320 in atopic dermatitis (AD) patients and 772 in those patients with both AD-ARD. The marked elevation of the serum IgE in the latter group was not associated with respiratory disease activity and not closely correlated with extent of the dermatitis, but may relate to patients with both AD-ARD being 'highly atopic'. Overall, there was no correlation between the serum IgE level and the frequency of positive skin tests. Compared to ARD patients, the serum of AD patients contained more IgE, yet they reacted significantly less frequently to common extrinsic allergens.


Antireticulin antibodies in dermatitis herpetiformis and adult coeliac disease. Their relationship to a gluten free diet and jejunal histology.

Antireticulin antibody was present in 17% of sera from thirty-five patients with dermatitis herpetiformis, 45% from twenty-eight patients with adult coeliac disease and none of thirty healthy controls. There was a higher incidence of antireticulin antibody(a) in patients on normal diets than in those on gluten free diets, and (b) in patients with subtotal villous atrophy than in those with partial villous atrophy. Two patterns of tissue staining were demonstrated, one of which was almost invariably associated with jejunal subtotal villous atrophy. The Ig class of the antibody was IgG in all instances but in 64% antibody of the IgA class was also present.


The erythemal action spectrum and deoxyribonucleic acid repair synthesis in xeroderma pigmentosum.

The reactions to monochromatic radiation of the skin of ten patients with xeroderma pigmentosum were investigated, and eight were abnormal. The abnormalities consisted of papular and vesicular reactions and delay in the development of the minimal erythema dose reaction with wavelengths principally in the 290-320 nm range. Three patients were too young for full action spectra to be obtained but of those patients in whom all wavelengths were tested only one showed a reaction to radiation above 320 nm and this was at 340 nm only. In only one patient was the minimal erythema dose at 300 nm at 24 h lower than normal. In six patients the repair synthesis of deoxyribonucleic acid after ultraviolet radiation was estimated. Reduced levels were seen in five but repair was normal in one patient. The patient with normal repair also had normal reactions to monochromatic radiation. The abnormal reaction of the skin to artificial radiation and the abnormal deoxyribonucleic acid repair synthesis may enable the diagnosis of xeroderma pigmentosum to be made at a very early age. In one of the patients the diagnosis was made at the age of 6 months in a child with photosensitivity but with no other clinical signs of the disease. It is suggested that, by making the diagnosis as early as possible and by protecting the skin from natural sunlight, cutaneous malignancies may be prevented.


Microvascular response to local application of dequalinium chloride. A vital microscopical study of hamster's cheek pouch and a microangiographic study of rabbit's ear.

Dequalinium chloride is a topical antiseptic, whose use may be attended by skin necrosis. Since microcirculatory changes are an early and distinct manifestation of tissue injury, the tissue damaging effect of dequalinium chloride on the microcirculation was examined. The immediate vascular reactions were studied by vital microscopy, of the hamster cheek pouch and the late effects were visualized by microangiography of the rabbit ear. An aqueous solution of dequalinium chloride in concentrations well below those used in clinical practice induced severe microcirculatory disturbances.


Plasma vitamin A levels in the hyskeratoses.

No significant difference was found between the plasma vitamin A levels of patients with dyskeratoti skin disease and those of comparable control subjects.


Treatment of solar keratoses with a 5-fluorouracil and salicylic acid varnish.

In an attempt to avoid the side-effects of treating solar keratoses with 5% 5-fluorouracil (5-FU) ointment, a new pharmacological varnish containing 5% 5-FU and 5-10% salicylic acid to collodium was tried. Twenty patients with such lesions were treated. A drop of the varnish was applied on each lesion every 3 weeks. Only one to five applications on facial lesions were necessary to obtain apparent cure in all patients. Lesions reappeared in four patients, but were cured after a second and similar treatment. The keratoses of the hands were more resistant and needed seven applications in one patient and nine in the other.


Lymphocyte transformation test in allergic contact nickel dermatitis.

Twenty-five patients with clinically proven, patch test positive nickel dermatitis were studied with the lymphocyte transformation test (LTT). Serial dilutions of NiSO4 were present in the lymphocyte culture. None of the controls showed lymphocyte blast transformation. In fourteen patients maximal incorporation of thymidine occurred at the highest dilutions (I:50,000-I:100,000). In eleven patients the highest incorporation occurred at the lower dilutions. The nature of the antigen responsible for the specific reaction remains unknown.


Anti-viral treatment of warts.

Several anti-DNA agents have been tested for their value in the treatment of warts in humans. A pilot study was carried out on patients with warts resistant to other forms of treatment. The agents used were hydroxyurea, idoxuridine and photodynamic inactivation. Idoxuridine 20% in a cream base appears to offer some possibilities as a treatment when other measures have failed.


The clinical spectrum of Sweet's syndrome (acute febrile neutrophilic dermatosis)-a report of eighteen cases.

Eighteen cases of Sweet's syndrome are described. This is the largest series so far reported and the first from the tropics. The essential features are the characteristic morphology, the histological appearances, the dramatic response to corticosteroids and the absence of scarring. Attention is drawn to the frequent involvement of the eyes and joints during the course of the illness. Fever and neutrophilia were found less frequently than a raised ESR or a preceding upper respiratory tract or skin infection. The term Sweet's syndrome is preferred to acute febrile neutrophilic dermatosis. The frequency of occurrence of different clinical manifestations is discussed.


Cell migration and DNA synthesis in organ culture of human skin.

Cell migration and DNA synthesis in organ  cultured skin were studied. Cell migration leads to the formation of a new epithelial layer around the free edges of the dermis. No significant DNA synthesis occurs in this newly formed epithelium. In contrast, DNA synthesis in the original epidermis is maintained throughout the period of culture as shown by the uptake of radioactive thymidine. Addition of hydrocortisone to the culture medium increases cell migration, and phytohaemagglutinin, while dramatically stimulating mitotic activity, suppresses cell migration. These results show that an increase in epithelial cell division is accompanied by diminished cell migration whereas an increase in cell migration occurs in the presence of a decrease in epithelial cell division. The behavaiour of skin in organ culture during the first 48-72 h can be compared with that seen in wound healing.


Smooth muscle antibodies and alopecia areata.

In seventy-eight patients with alopecia areata, the incidence of auto-antibodies to smooth muscle was greater than that in an age and sex matched control population.


Serum anti-tumour antibodies and auto-antibodies in vitiligo.

Anti-tumour antibody was sought in the sera of three groups of patients; idiopathic vitiligo, malignant melanoma and miscellaneous skin disorders. No precipitating antibody to a series of prepared pigmented tumour antigens was demonstrated. All sera were tested for a number of auto-antibodies. No significant differences were detected between the groups. The evidence for an immunological factor in the causation of vitiligo remains circumstantial only.


The modifying effect of autologous serum on leukocyte migration inhibition by liver antigens in systemic lupus erythematosus.

The leukocyte migration test was used to examine patients with systemic lupus erythematosus (SLE) for evidence of cell-mediated immunity to human liver microsomes and mitochondria. Testing of both these antigens with leukocytes from patients was carried out simultaneously in culture medium enriched by either 20% fetal bovine serum (the standard test) or substituted 20% autologous serum. Normal controls showed little or no reactivity in the standard test whereas the SLE group revealed a high incidence of significant leukocyte migration inhibition to both antigens which, however, was considerably reduced by the substitution of autologous serum. The results suggest that the sera of patients with SLE contain factor(s) capable of blocking the in vitro expression of cell-mediated immunity to human tissue antigens. These factor(s), if active in vivo, might have a role in controlling some of the auto-immune manfestations of the disease.


Emotions and skin (II)-the conditioning of scratch responses in cases of lichen simplex.

Lichen simplex is generally regarded as a condition initiated and perpetuated by scratching and emotional tension. It was felt that the scratching might partly be a conditional response to itching and other signals, and that feelings of guilt, anxiety and hostility would be prominent features in these patients. Conditioning experiments designed to establish scratch responses to an itch stimulus (UCS) and a tone (CS) showed that lichen simplex patients conditioned more readily and extinguished more slowly than controls. These finding were more marked when the itch stimulus was applied to affected as compared with normal skin. It was not possible to distinguish differences in the psychological tests between patients and controls. The possible significance of these findings is discussed.


The areal extent of dermatophyte infection.

After stripping with vinyl tape, dermatophytes may be cultured throughout the depth of the stratum corneum. In the present study skin strippings were used to investigate the horizontal spread of the dermatophyte infection. In five cases of tinea cruris and in twenty-two cases of tinea corporis, fungi could be cultured not only from the visible lesion, but in the majority of cases also from the normal-looking skin up to 6 cm from the margin of the lesion. No relationship could be found between the size and age of the lesion and the invasion of surrounding, normal-looking skin.


A study of chemical light screening agents.

In a group of 104 non-photosensitive subjects and seventy-eight suffering from a variety of photodermatoses, the most effective protective preparation against irradiation by short UV sunburn wavelengths (smaller than 320 nm) was found to be a mixture of 5% para-aminobenzoic acid and 2.5% of the ester amyl dimethylaminobenzoate in 70% ethanol with para-aminobenzoic acid being usually more effective than the ester, and mexenone the least effective individually. The protective capacity was found to be reduced by showering but not by exercise. The addition of glycerol to the formulations did not affect their protective capacity although the addition of propylene glycol did increase the protection obtained.


The HL-A system and psoriasis.

A study of 157 psoriatic patients showed that 48.4% were HL-A 17 positive (control 6.0%). The relative risk of psoriasis in HL-A 17 positive individuals (14.7) was significantly higher than that reported from other centes, whilst that for HL-A 13 was lower. HL-A 17 was commoner in femal patients and associated with an earlier age of onset of psoriasis than when HL-A 17 was absent. The earlier age of onset in females is largely due to the higher frequency of HL-A 17. Family studies show that often the ingeritance of HL-A 17 is a necessary condition for psoriasis to appear and this probably applies in some instances to HL-A 13. It is likely that there is interaction between HL-A 17 and HL-A 13 and other genes predisposing to psoriasis. In some families there appears to be no association between psoriasis and the HL-A system. The general significance of these findings is discussed.


The age-of-onset of psoriasis and the relationship to parental psoriasis.

In series of 419 consecutive patients with psoriasis the peak age-of-onset was between 5 and 9 years of age in girls and between 15 and 20 years in boys. Subsequently, there was a gradual decline in relation to age. The incidence of psoriasis in a parent of a proband was shown to be significantly higher in those patients where a detailed exmination of the family was undertaken, as compared with the information obtained only from case records.


M. fortuitum and M. chelonei-fast growing mycobacteria. A review with a case report.

Infection due to opportunist mycobacteria is considered in general and that due to the fast growing mycobacteria in particular. The group is now seen to contain two facultative pathogens, M. fortuitum and M. chelonei (abscessus), all other synonyms for these two species having been shown to be invalid. Both organisms are ubiquitous and normally harmless; however, following injury or injection, they may cause chronic sub-cutaneous abscesses. Pulmonary lesions have also been described. More recently M. fortuitum has been found to cause osteomyelitis and corneal ulceration and both organisms have been identified in cases of septicaemia associated with renal dialysis. In the past M. chelonei my have been identified as M. fortuitum but although it is more difficult to isolate, it may in fact be the more common cause of disease. A case is reported of an English girl who received a small injury to her shin in Spain. The initial lesion failed to heal and for 18 months there was repeated formation small sinuses in the area. Healing then occurred spontaneously. Histology showed a chronic granuloma and on two occasions a rapid growing mycobacteria resistant to all antibiotics was isolated. This was initially identified as M. fortuitum but finally as M. chelonei and the latter diagnosis was confirmed by differential skin tests. Difficulties in interpretation of these tests are discussed.


Wegener's granulomatosis-controlled by azathioprine and corticosteroids.

A case of Wegener's granulomatosis is presented with control of lung lesions by corticosteriods and renal lesions by azathioprine. It supports previous experience of antimitotic therapy in this condition.


Anonychia with bizarre flexural pigmentation-an autosomal dominal dermatosis.

A nulliparous female aged 26 years, who presented with long-standing sore and cracked soles, showed congenital anonychia, long-standing hyper-and-hypo-pigmentation in axillae and groins, dry palmar and plantar skin as well as other skin and hair abnormalities. She gave a family history of nail, hair and pigmentary abnormalities and examination of living affected relatives confirmed this. This appears to be a hitherto undescribed autosomal dominant syndrome.


Comparison of in vivo effect of inorganic lead and cadmium on glutathione reductase system and delta-aminolevulinate dehydratase in human erythrocytes.

The activity of delta-aminolevulinate dehydratase (ALAD) of erythrocytes, the lead (Pb-B) and cadmium (Cd-B) concentration in whole blood, the content of reduced glutathion (GSH) in erythrocytes, and the regeneration rate of GSH by intact erythrocytes were measured during an epidemiological survey of 84 men employed in a Belgian cadmium and lead producing plant. A control group of 26 persons (students and laboratory staff) was also examined. The logarithm of the ALAD activity is highly inversely correlated with log Pb-B (r = -0.760) but no correlation was found with log Cd-B. There exists a significant negative correlation between GSH and log Pb-B (r = -0.423) but not between GSH AND LOG Cd-B. The apparently good relationship between log ALAD and GSH disappeared completely by holding log Pb-B constant, but log ALAD remained highly inversely correlated with log Pb-B when standardized for GSH concentration (r = -0.748). In vivo investigation of the GSH regeneration rate of intact erythrocytes demonstrated clearly that the overall activity of the glutathione oxidation-reduction pathways is not impaired in Pb and Cd-exposed workers with significantly increased Pb-B and Cd-B, since their initial GSH regeneration rate (first 15 minutes) was identical with that of the control group. Results of similar in vitro experiments in which control whole blood was incubated before-hand with Pb2+ or Cd2+, or both, reinforce this conclusion. Since increased Cd-B and Pb-B do not influence the glutathione reductase system of erythrocytes, and since endogenous erythrocyte GSH is not correlated with Cd-B, the moderate decrease in endogenous erythrocyte Gsh found in Pb-exposed workers might result from a Pb-induced impairment for the erythrocyte mechanism for glutathione synthesis.


Absenteeism among survivors of the atomic bombing of Hiroshima.

Atomic bomb survivors who worked at the Atomic Bomb Casualty Commission in Hiroshima during the years 1968-71 and held handbooks identifying them as survivors took significantly more days of both annual leave and sick leave than did matched and paired control subjects. These differences in leave-taking patterns are considered to be due to behavioural causes as they could not be attributed to radiation dose-response effects.


An epidemiological study of occupational bladder tumours in the dye industry of Japan.

This report studies 100 cases of occupational bladder cancer which were reported between 1949 and 1970 by large companies producing dye. It is estimated that in fact a great many more than 100 cases may have developed during the 56 years from 1914 to 1970, as this period corresponds with the beginning of dye production to the termination of benzidine and beta-naphthylamine manufacture in Japan. New cases of occupational bladder tumour should diminish significantly in the future in Japan on account of the prohibition of the production of beta-naphthylamine and benzidine. However, it is important to follow-up continuously workers who have been exposed to these amines. In Japan no cases of occupational bladder tumours from the rubber industry have been reported. It is emphasized that prospective epidemiological studies are required not only in the rubber industry but also in other industries where amines may constitute an occupational hazard.


Chronic cyanide exposure: a clinical, radioisotope, and laboratory study.

The effect of chronic cyanide exposure in the electroplating sections of three factories employing 36 workers was studied and compared with a control group. The concentration of cyanides to which the workers were exposed was measured. The regression line showing the relationship between thiocyanates in urine and the concentration of cyanides in the air was plotted. Increased percentages of haemoglobin and lymphocyte count were present in all exposed workers, in addition to punctate basophilia in 28 workers. Cyanmethaemoglobin was found to be characteristic. Apart from other complaints, two men with psychosis similar to one case reported in therapeutic thiocyanate intoxication were found. Twenty of the workers had thyroid enlargements to a variable degree and consistency, in two of whom it resembled lymphadenoid goitre. Thyroid 131I uptakes at 4 and 24 hours were significantly higher than in the controls, while 131PBI was unchanged. The reason for this iodine deficiency-like action is discussed.


The ventilatory cost of activity.

The energy cost of activity, depending on its intensity, varies between 6 and 50 kJ min-1 with average values for essential daily tasks and for hard work respectively of 12 and 28 kJ min-1. The levels of ventilation required to sustain these activities are, on average, 15 and 33 1 min-1 with an overall range of +/- 30%. Additional ventilation is needed for speech. The requirements for individual activities are reviewed.


Chest symptoms in farming communities with special reference to farmer's lung.

Surveys were carried out on random samples of the farming population in Devon and Wales in order to estimate the prevalence of respiratory symptoms and of positive precipitin reactions to thermophilic fungi. Bronchitis, as defined, was common among the Welsh hill farmers, and the proportion of positive serological tests was higher in both the areas surveyed in Wales as compared with Devon. All three surveys confirmed a previous finding that the proportion of positive precipitin tests was higher among non-smokers than smokers. Although the numbers were small there was some indication that measurement of peak expiratory flow showed different relationships with age in non-smokers according to the presence or absence of positive precipitin tests. The difficulty of determining prevalence rates for farmer's lung is discussed, but the results suggest a rate not dissimilar to those found in two areas of Scotland which were more than 20 times higher than any figure previously reported in Britain.


Pilot study of closing volume in byssinosis.

A study of the relative sensitivities of forced expiratory volume in one second (FEV1), maximal mid-expiratory flow (MMF), and closing volume (CV) in the detection of subjects with byssinosis was carried out in a North Carolina cotton mill. Altogether 35 workers participated in the study. Of these, nine showed a decline in FEV1 of 10% or more during the first work shift that followed the weekend break. Twelve subjects showed a decrease in MMF of 15% or more. In contrast only six workers exhibited a 10% increase in closing capacity, while ten showed a 10% increase in CV. Recent evidence of the magnitude of variability in closing volume manoeuvres suggests that our chosen level of change was too low, A 40% change in CV would have identified only five subjects. CV is a more complex manoeuvre for the subject being tested and for the technician to perform, is more time consuming, and is subject to greater variation. To have any advantage over spirometry, CV would have to be appreciably more sensitive. Our study suggests that it is not. However, the MMF may prove to be more sensitive than the FEV1 in the detection of byssinosis.


An evaluation of effect of airborne dust from a cotton mill on the guinea-pig ileum with reference to byssinosis.

The effect of airborne dust on the guinea-pig ileum was studied. Tyrode extracts of airborne dust collected freshly in the cardroom of a cotton mill, and extracts of air pollutant samples drawn on the roof of the mill and of the local town hall were all found to induce the guinea-pig ileum to contract when applied in a tissue-bath. However, the force of contraction with air pollutants was rather greater than that with the cardroom dust. Considering the variables involved, the ileum response to the cardroom dust may have been due to ordinary air pollutants which constitute a significant part of the dust. It is concluded that this pharmacological phenomenon is probably not relevant in the context of byssinosis.


Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isozymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi.

The extracellular fluid of the plant pathogen, Fusarium solani f. pisi, grown on the plant cuticular polymer, cutin, was shown to contain cutinase and p-nitrophenyl palmitate hydrolase activities (R.E. Purdy and P.E. Kolattukudy (1973), Arch. Biochem. Biophys. 159, 61). From this extracellular fluid two isozymes of cutinase and a nonspecific esterase (p-nitrophenyl palmitate hydrolase) were isolated using Sephedex G-100 gel filtration, QAE-Sephadex chromatography, and SE-Sephedex chromatography. Phenolics contained in the extracellular fluid were found to be associated with the cutinase but not with the nonspecific esterase, and the phenolic materials were removed from cutinase at the QAE-Sephedex step. A 34-fold purification of the nonspecific esterase and a 6.5-fold purification of cutinase were achieved by the procedure described. The two isozymes of cutinase (I and II) and the nonspecific esterase were homogeneous as judged by polyacrylamide disc gel electrophoresis and sedimentation equilibrium centrifugation. Molecular weights of cutinase I, cutinase II, and the nonspecific esterase were determined by Sephedex G-100 gel filtration, sedimentation equilibrium centrifugation, amino acid composition, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The values obtained with these techniques agreed with each other and were about 22,000 for both cutinases and 52,000 for the nonspecific esterase. The dodecyl sulfate gel electrophoresis indicated that a small portion of cutinase II contained proteolylic clips, near the middle of the polypeptide chain, and that the nonspecific esterase might also have undergone some proteolylic modification. The amino acid composition of cutinase I was similar to that of cutinase II except for the presence of a larger number of tryptophan residues in the latter, while the amino acid composition of the nonspecific esterase showed more differences from that of either cutinase.


Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase.

The stereochemistry of the two half-reactions catalyzed by the biotin-containing enzyme, transcarboxy-lase from Propionobacteria shermanii, has been determined. The pro-R hydrogen at C-2 of propionyl-coenzyme A is replaced by CO2 in formation of the S isomer of methylmalonyl-CoA, defining the process as retention of configuration. This C-2 hydrogen is abstracted at a rate identical with product formation. For the other half-reaction, pyruvate to oxalacetate, the chiral methyl group methodology of Rose (I. A. Rose (1970), J. Biol. Chem. 245, 6052) was employed. First, it was determined with [3-2-He]pyruvate that a kinetic deuterium isotope effect of 2.1 occurs at Vmax in this carboxyl transfer, indicating that the necessary requirement for discrimination against heavy isotopes of hydrogen existed. Then, 3(S)-[3-2-H,3-H]pyruvate, generated from 3(S)-]E-2-H,3-H]phosphoglycerate, was carboxylated and the oxalacetate trapped as [3030H]malate using malate dehydrogenase. Exhaustive incubation of the tritiated malate (3-H/14-C = 1.95) with fumarase to labilize the pro-R hydrogen at C-3 resulted in release of 65% of the tritium into water. Reisolation of the malate after fumarase action yielded a 30H/14-C ration of 0.67, indicating 34% retention as expected. The theoretical enantiotopic distribution for the observed k1H/k2H of 2.1 is 68:32. Selective enrichment of tritium in the pro-R position at C-3 of malate indicates enzymatic carboxylation of pyruvate with retention of configuration in this half-reaction also.


Biosynthesis of the polyoxins, nucleoside peptide antibiotics: a new metabolic role for L-isoleucine as a precursor for 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid).

The biosynthetic origin of the carbon skeleton of 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid) is described. This unique cyclic amino acid is the C terminus of the nucleoside peptide antibiotics, the polyoxins, elaborated by Streptomyces cacaoi var, asoensis. In vivo experiments show that 14-C from [1-14-C]isoleucine, [U-14-C]isoleucine, [1-14-C]methionine, [U-14-C]methionine, [U-14-C]threonine, and [1-14-C]glutamate is incorporated into polyoximic acid; however, 14-C from [5-14-C]glutamate and [methyl-14-C]methionine is not incorporated. The distribution of 14-C in polyoximic acid clearly shows that the intact carbon skeleton of L-isoleucine is utilized directly. The incorporation of 14-C from [U-14-C]methionine, [U-14-C]threonine, and [1-14-CA1glutamate into polyoximic acid occurred only after their conversion to isoleucine via 2-ketobutyrate. A scheme is presented in which either of the two beta-unsaturated amino acids isolated from Bankera fuligineoalba, L-2-amino-3-hydroxymethyl-3-pentenoic acid or L-2-amino-3-formyl-3-penetenoic acid, is regarded as a possible intermediate amino acid between isoleucine and polyoximic acid.


The structure of kinetoplast DNA. I. Properties of the intact multi-circular complex from Crithidia luciliae.

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix  density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.


Electrode measurement of oxygen tension with 1-ms time resolution.

A continuous flow device utilizing a Clark oxygen electrode was constructed; this device had a dead time and resolution of 1 ms. Mixing was tested by observing the neurtralization of acid with base, and at the maximal flow rate, the mixing was 94% complete within 1 ms and better than 98% complete within 2 ms after initial mixing. Observation o of the oxygenation of hemoglobin gave data which agreed with previous data obtained by a stopped-flow optical experiment. The respiration of phosphorylating submitochondrial particles was measured utilizing this device. The burst of respiration in submitochondrial particles was triphasic, with a very rapid burst lasting some 60 ms, followed by a longer burst of respiration lasting more than 4 s.


Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores.

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, D-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.


Immunological quantitation of chloroplast ferredoxin.

An immunosassy for the quantitative determinations of ferredoxins in cell free extracts from plant tissues is described. The method is accurate for the assay of 0.3-1.5 nmol ferredoxin directly from the extracts. The following average values (nmol ferredoxin/mg extractable protein) were obtained: 3.9, 1.8. 5.90, 14.8 and 10.9 for Euglena gracilis, spinach, parsley, lettuce, and broccoli, respectively. Specific factors affecting the method are discussed in detail.


Light-induced turnover of chloroplast cytochrome b-559 in the presence of N-methylphenazonium methosulphate.

In the presence of 0.1-5 muM N-methylphenazonium methosulphate approx. 50-70% oxidation of cytochrome b-559 can be induced by far-red light. The oxidation is best observed with long wavelength far-red light (732 nm) of moderate intensities (approx. 10(4) ergs/cm2 per s) and is reversed by subsequent illumination with red light. Concentrations of N-methylphenazonium methosulphate above 5 muM are inhibitory probably due to cyclic electron flow. The far-red oxidation is inhibited by low concentrations of the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibits red light reduction and increases the amplitude of far-red oxidation. The effect of N-methylphenazonium methosulphate is mimicked by N-methyl-phenazonium ethosulphate, but not by pyocyanine or diaminodurene. Low concentrations (2-3 muM) of N-methylphenazonium methosulphate also stimulate a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-inhibitable red light reduction of cytochrome f.


Insulin degradation. XV. Use of different assay methods for the study of mechanism of action of glutathione-insulin transhydrogenase.

Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 125 I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Line-weaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent Km values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.


Palmitic acid activation of peroxidase and its possible significance in mango ripening.

Palmitic acid stimulated the activity of mango peroxidase and reversed the inhibition due to the peroxidase inhibitor present in the preclimacteric fruit. The palmitic acid effect appeared to saturate in the range of 45 to 60 muM palmitic acid. Crude fatty acid extract of the mango exerted similar effect. The percentage stimulation was pH-dependent. Palmitic acid stimulated the enzyme by 18 percent at its optimum pH (5) but the stimulation was in excess of 63 percent at pH 2.5. At pH 2.5 the enzyme concentration versus velocity plot was non-linear and the activation by palmitic acid appeared to saturate between 32 and 48 muM concentration of the effector. The inhibition of the enzyme at and above 0.86 muM concentration of substrate (H202) was not found in the presence of palmitic acid. The effector also changed the heat inactivation kinetics of the enzyme and activated only two out of the four peroxidase isoenzymes present in the climacteric fruit extracts. The results presented indicate the regulatory nature of the enzyme and support its significance in fruit ripening.


Further characterization of L-leucine-pyruvate transaminase from Acetobacter suboxydans.

L-Leucine-pyruvate transaminase obtained from Acetobacter suboxydans exhibited absorbance maxima to 280 and 332 nm. The 332 nm peak was derived from the coenzyme bound to the enzyme protein with the epsilon NH2 of a lysine residue. The transaminase showed reactivity against many L-amino acids. The relation between the reactivity and the structure of the amino donor is discussed. The Michaelis constants for L-leucine, pyruvate, L-alanine and alpha-ketoisocaproate were 6.7, 3.1, 7.1 and 0.9 mM, respectively. The equilibrium constant was 5.3. The activation energy at pH 5.0 was 8,800 cal/mol.


Fractionation of the rat liver enzymes that hydrolyze benzoyl-arginine-2-naphthylamide.

1. The enzyme activity in the particulate fraction from rat liver that hydrolyzes alpha-N-benzoyl-DL-arginine-2-naphthylamide (Bz-Arg-NNap) has been separated into two approximately equal components by chromatography on DEAE-cellulose. One component (peak II) is completely retained by the column at low ionic strength while the other component (peak I) passes through. 2. In contrast to the enzyme in peak I, the enzyme in peak II is extremely sensitive to inhibition by leupeptin, it will hydrolyze carbobenzoxy-alanylarginylarginyl-4-methoxy-2-naphthylamine, and it will inactivate aldolase. 3. There appears to be also a minor high molecular weight component of the alpha-N-benzoyl-DL-arginyl-2-naphthylamine-hydrolyzing activity that is retained by the DEAE-cellulose but which has properties similar to those of the peak I enzyme.


Physicochemical and kinetic properties of beef liver argininosuccinase. Studies in the presence and absence of arginase.

Physicochemical properties of beef liver argininosuccinase (EC 4.3.2.1) are reported, which show that the operational kinetic unit at pH 7.5 in the concentration range used for enzymic assays is of molecular weight 203 000. This information is utilized in the interpretation of initial velocity studies of the forward reaction (argininosuccinate as initial substrate) where, in an Eadie-Hofstee plot, deviation from linearity is observed. These results are examined with the aid of a rate equation, formulated as a ratio of two polynomials in initial substrate concentration, which provides a quantitative description of the system in terms of cooperative effects. The kinetics of the reverse reaction catalysed by argininosuccinase were also investigated by performing experiments of different design in which the rate of change of fumarate concentration was monitored as a function of time. Comparison of these results with numerical solutions obtained by integrating the differential rate equation reflecting the reversible reaction permitted estimation of the relevant kinetic parameters. One of the latter experiments involved a coupled assay with argininosuccinase and arginase as the consecutive catalysts and indicated that no chemical interaction occurred between the enzymes. This observation, which is relevant to consideration of fluxes in the urea cycle, was supported by both sedimentation velocity studies on mixtures of the enzymes and by other kinetic analysis.


Mammalian 3-oxosteroid delta4-delta5-isomerase: a membrane-bound enzyme. I. Fluorescence study of the relationship between the enzymatic binding site, phospholipids, water and ions.

The microsomal membranes and the proteolipidic particles obtained by disruption of the microsomes by alkaline-earth ions at molar concentration have been compared by measuring the fluorescence properties of 1-anilino-naphthalene-3-sulfonate and naphthyl-1-phenylamine. The protein lipid arrangement of these two systems appears to be not essentially different. The study of fluorescence polarization of an hydrophobic probe (perylene) in function of Mg2+ concentration suggests a possible mechanism of disruption of the membrane by Mg2+ involving the strong structure-making effect of the ion. The comparison of the fluorescence polarization changes of perylene and equilenine (a competitive inhibitor of the isomerase) with the ionic concentration indicates that there is no direct relation between the bulk lipidic phase and the enzymatic binding site properties. Moreover, the emission of equilenine is completely quenched by I-, in contrast with the napththyl-1-phenylamine and perylene probes, which clearly demonstrates the accessibility of the catalytic site to water molecules and ions.


Mammalian 3-oxosteroid delta4-delta5-isomerase. A membrane-bound enzyme. II. Activation by divalent cations.

Alkaline-earth ions (Mg2+, Ca2+ and Sr2+) have two specific effects on the kinetic parameters of the beef adrenal 3-oxosteroid delta4-delta5-isomerase activity in the microsomes and in the particles obtained after disrupting the membrane structure by action of 1 M MgCl2. On the microsomal enzyme, a 2-fold increase of V is observed with the three cations under study. The small difference in the effect of the three ions could be related to their hydration energy. It is suggested that the interaction of the ion with water is the determinant step of the activation mechanism and not the fixation of the ion on the enzyme or on some others possible binding sites in this system. With the enzyme in the proteolipidic particles, the use of EDTA as a chelating agent for the cations present in the enzymatic assay, allows the characterization of two effects: at low concentration of EDTA, an increase of Km is observed and at higher concentration (2 mM), V is decreased. A subsequent addition of Mg2+ leads to an activation in two steps: V is increased in the first step without change in Km, the second step consists of a decrease of Km without any change in V. A relation between the structural perturbations induced by the ions (Gallay, J., Vincent, M. and Alfsen, A. (1975) Biochim. Biophys. Acta 397, 489-500) and their kinetic effect on the enzymatic reaction is established.


Structural requirements for the enzymatic phosphorylation of phosvitin.

Some structural features required for the enzymatic phosphorylation of phosvitin by purified rat liver cytosol phosvitin kinase have been investigated by testing the activity of such an enzyme toward phosphopeptides differing in size and chemical composition, obtained by pronase or acid hydrolysis of phosvitin. The results obtained can be summarized as follows: (a) Phosvitin kinase phosphorylates even fairly simple phosphopeptides (mol.wt 1000-2000) at rates comparable with intact phosvitin. (b) Acetylation of both phosvitin and pronase phosphopeptides completely prevents their phosphorylation indicating that some lysine residues are strictly required for the phosvitin kinase reaction. (c) Accordingly polyphosphorylserine blocks Ser(P)n which are very actively phosphorylated in phosvitin and pronase phosphopeptides, do not undergo any more enzymatic phosphorylation once isolated as such in a form free of other amino acids. (d) The activity of phosvitin kinase toward substrates probably devoid of Ser(P)n blocks suggests that there are not required for the protein kinase reaction. However, they apparently enhance the phosphorylation rate of the peptide substrates, likely by making easier their binding to the enzyme. It is proposed therefore that the peptidic unit able to undergo phosphorylation by rat liver cytosol phosvitin kinase consists of one or more phosphorylserine residues having in their close proximity a lysine residue playing a critical role in the mechanism of transphosphorylation.


Lateral diffusion of cholesterol in monolayers.

The surface diffusion coefficient of cholesterol in cholesterol monolayers has been measured as a function of cholesterol surface concentration. Two different radiochemical methods, one integral and the other differential, were developed which gave comparable results. In the integral method two cholesterol monolayers, one of which is radioactive, are isolated on inert hydrophilic supports and then brought into contact. After some time the supports are separated and the radioactivity of the supports is measured. The differential method is an autoradiographic experiment. Two cholesterol monolayers, one of which is radioactive, are separated by means of a thin barrier. Upon removal of the barrier and at later times, an autoradiographic plate is brought to within a fraction of a mm from the aqueous surface and exposed. The plates are developed and analysed. The data show that the cholesterol surface diffusion coefficient in the dilute monolayers is approximately 10(-6)cm2/s and is nearly independent of surface concentration up to a concentration corresponding to an area of 40 A2/molecule. As the monolayer becomes compressed beyond this surface concentration, the diffusion coefficient decreases ubruptly with the deeply decreasing surface tension to about 10(-7) cm2/s, when a fully condensed surface layer of 38 A2/molecule is reached. This diffusion coefficient is of the same order of magnitude as the diffusion coefficients measured in lipid bilayers and in membranes.


Carotenoids as a Raman-active probes of erythrocyte membrane structure.

1. Erythrocyte ghosts exhibit resonance-enhanced Raman bands at 1530 cm(-1) and 1165 cm(-1) attributable to v(-C=C-) and v(=C-C=), respectively, of the conjugated polyene chains in carotenoids. In lipid extract of ghosts, these resonance-enhanced bands lie at 1527 and 1158 cm(-1). The spectra indicate the presence of membrane-bound beta-carotene. 2. The resonance-enhanced Raman spectrum of beta-carotene in lecithin liposomes is identical to that obtained with hexane or chloroform solutions. 3. Increasing proportions of cholesterol in cholesterol-lecithin liposomes up to a cholesterol: phospholipid molar ratio of 0.8-0.9 drastically decreases the intensity of both resonance-enhanced bands. 4. In ghosts the carotenoid bands respond to membrane perturbations. Trypsinization, lysolecithin treatment and reduction of pH increase the intensities of the 1530 and 1165 cm(-1) bands. In contrast, a decrease in the intensity of both bands follows equilibration of ghosts for 15 min at approx. 50 degrees C or addition of (0.1%) sodium dodecyl sulfate. 5. We suggest that perturbants known to change lipid-protein interactions in erythrocyte membranes modify the microenvironment and/or configuration of the membrane-bound carotenoid.


Lipid-protein interactions at the erythrocyte membrane. Different influence of glucose and sorbose on membrane lipid transition.

When observed over a temperature range, erythrocyte membrane lipids undergo a transition at 18-20 degrees C (Zimmer, G. and Schirmer, H. (1974) biochim. Biophys. Acta 345, 314-320). This observation has prompted an investigation of the effects that substrate binding has on the transition of the red cell membrane. Glucose and sorbose were compared, since transport kinetics of these sugars still pose unresolved questions. In membranes, preloaded with glucose, the break at the transition temperature was intensified, while it was abolished or reversed in membranes preloaded with sorbose. These results were corroborated using different solubilization procedures (sonication, sodium dodecyl sulfate treatment) of the membranes, and also different techniques (viscosimetry, 90 degrees light scattering, 1-anilino-naphthalene-8-sulfonate fluorescence). In extracted membrane lipids, viscosimetry indicated a break at transition temperature after preloading with either glucose or sorbose. Disc electrophoresis revealed a different binding pattern of the two sugars. It is suggested, that the amplification of the discontinuity in red cell membranes by glucose and the abolition or reversal of the break by sorbose are mediated by membrane protein- and/or membrane lipid-protein interaction.


The roles of bile salts in the uptake of beta-carotene and retinol by rat everted gut sacs.

The effects of bile salts, Tween 20 and hexadecyltrimethylammonium-bromide on the uptake of beta-[3H]carotene and [3H]retinol by rat-everted gut sacs were studied in vitro under conditions simulating those present in the intestinal lumen during lipid absorption. 2. Micellar solutions significantly enhanced uptake over emulsions. Maximum uptake occurred at the critical micellar concentration of the bile salts mixture. At higher detergent concentrations beta-carotene uptake declined sharply; retinol absorption remained high. 3. In beta-carotene absorption bile salts functioned not only as micellar solubilizers but also may have been required for interaction with the cell membrane or as a transport carrier. In retinol uptake their primary function appeared only to be micellar solubilization. Both uptake and efflux of substrates were enhanced in bile salt micellar solutions compared to the other detergents. 4. Beta-carotene cleavage and conversion to retinyl esters occurred only in bile salts solutions. Retinol esterification was seen with all detergents. These effects increased as the tri/dihydroxy bile salts ratio was increased. 5. Beta-carotene uptake appeared to be reversible and passive at low concentrations. Retinol uptake was reversible, 7-30 times more rapid, and partially inhibited by 2,4-dinitrophenol at higher concentrations. An energy-requiring step may have been rate limiting.


Amino acid transport in placental slices. Mechanism of increased accumulation by prolonged incubation.

The accumulation of alpha-aminoisobutyric acid by placental slices is increased dramatically upon prior incubation of the slices in amino acid-free, buffered saline. This increase is inhibited by inhibitors of protein synthesis and is accompanied by an increased V for the transport process. While alternative explanations are discussed, these data suggest that the incubation effect may be mediated through an increase in the number of available transport sites which are synthesized during the incubation period. Incubation with an amino acid mixture diminishes the increase as well as general protein synthesis, suggesting that a reduced availability of amino acids may initiate compensatory changes in the synthesis of cellular transport proteins.


The site of metal ion binding in a nickel derivative of adenosine 5'-monophosphate: an x-ray study.

Single-crystal X-ray methods have been used to characterize a nickel derivative of adenosine 5'-monophosphate, of stoicheiometry [Ni(5'-AMP)(H2O)5]-H2O. The metal atom binds to the N(7) position on the adenine base, with the five remaining octahedral coordination sites about nickel occupied by water molecules. The phosphate group is connected via intramolecular hydrogen bonds to coordinated water molecules.


Cellular site of synthesis of ribosomal proteins in yeast.

Looking for messenger RNA coding for yeast ribosomal protein, we devised a method to identify polysomes involved in ribosomal protein synthesis. Analysis of nascent protein elongated in vitro demonstrated that ribosomal proteins are synthesized both on membrane-associated and free polysomes.


Albumin synthesis and catabolism following partial hepatectomy in the rat. The effects of amino acids and adrenocortical steroids on albumin synthesis after partial hepatectomy.

Plasma albumin levels were measured in partially hepatectomized, sham operated and control rats. The levels fell in both the partially hepatectomized and sham operated groups; while the latter group returned to normal within a few days, the low plasma albumin in the partially hepatectomized animals was sustained. Albumin synthesis rates in the isolated perfused rat liver were measured in the three groups of animals at varying intervals after partial hepatectomy. There was a significant depression of albumin synthesis rate in terms of both liver and whole animal weights when compared to the sham operated and control animals. This depression was almost completely reversed by the addition of arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine added together to 10 times their normal plasma concentrations. The addition of hydrocortisone had no effect on the albumin synthesis rate after partial hepatectomy. Studies in vivo in the three groups of animals (partially hepatectomized, sham operated and control animals) revealed a fall in the albumin catabolic rate after partial hepatectomy coinciding with the fall in the albumin synthesis rate. An hypothesis whereby the amino acids may have their stimulatory effect is proposed.


The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.


Selective attention and evoked potentials in humans--a critical review.

Human evoked-potential research on the neurophysiological substrate of selective attention is reviewed. Most of these studies report enhanced amplitudes of potentials evoked by attended (task-relevant, meaningful, important, etc.) stimuli the results of which are generally regarded as providing an electrophysiological correlate for selective attention. In accepting such claims, there appears to be two major procedural problems generally not satisfactorily solved in these studies: (1) the inability to reliably separate the specific and non-specific physiological changes concomitant with selective attention from each other; and (2) inadequacy of peripheral sensory control possibly inducing contaminating changes already at the level of the proximal stimulus. Problem (1) originates from, and the importance of (2) is emphasized by, the temporal stimulus structure of experimental tasks in these studies which allows the subject to predict above the chance level the relevant events and, thus, to differentially prepare himself for these in advance (increased non-specific arousal and selective peripheral sensory orientation, the latter often made possible by insufficient control, have possibly been among these changes). Those studies to which these two (and other) remarks do not apply at all or only to an insignificant degree have generally shown no selective evoked-potential changes (or these changes have occurred only with a long latency ('P3' or 'P300') making their interpretation especially uncertain). There is one exception for this general notion, the reasons for and significance of which are dealt with in detail. Finally, the difficulties and inherent limitations of inferring brain events from scalp-recorded evoked-potential data, especially with respect to the important selective-filter hypothesis of selective attention, are extensively discussed and, in the light of these difficulties, some trends for future research proposed.


Decreased skin conductance response habituation in chronically anxious patients.

Skin conductance response (SCR) habituation to loud tones was tested in chronically anxious patients and controls matched for age, sex and occupation. The results replicate Lader and Wing's finding of slowed response habituation in the patient group. These results are discussed in light of the Lader and Wing model attributing anxiety-proneness to an innate slowness in habituating to stimulation. Two factors which in themselves could cause slowed response habituation were present in the patient group during testing: increased physiological activity and increased threat appraisal. The latter resulted in the patients' frequently attributing signal value to the irrelevant tones. Because of the presence of these contaminating factors, and decreased response habituation in the patient groups can be considered only weak evidence in support of the slowed habituation model. Since the excessive test anxiety in the patient group contributes to both increased physiological activity and increased threat appraisal, it is suggested that SCR habituation testing be undertaken when both patient and control groups can be brought to equivalent states of relaxation.


A plasma protein mobilizing factor in dogs following hemorrhage.

Small amounts of whole blood from repeatedly bled donors increased the replacement rate of plasma protein in dogs, cats and rabbits subjected to hemorrhage. Results obtained within three hours were statistically significant. This strongly suggests that a chemical mediator, to hasten restoration of plasma protein levels, is released into the blood during hypoproteinemia.


[Lymphocyte depletion through experimental anastomosis of the thoracic duct and cervical esophagus].

In 25 dogs, a short segment of the left subclavian vein, adjacent to the connection of the thoracic duct, was dissected free with its jugular branch and ligated. An end-to-side anastomosis, using interrupted sutures was performed in two layers, between the side of the cervical esophagus and the proximal part of the jugular vein. This vein, acting as a reservoir, remains connected to the thoracic duct by the isolated segment of the subclavian vein. The original aspect, of our technique is the use of a catheter placed into a branch of the vein near the site of the anastomosis and through which a saline solution with heparin (1 mg/kg) and penicillin (40.000 U/kg) is injected daily. The catheter emerged on top of the dog's neck, and continuous perfusion was provided by a chronofusor. The catheter was removed after one month. In 30% of the dogs, the anastomosis was found patent after more than 6 weeks. This has been proved by fibro-esophagoscopy after injection of a solution of patent blue in the jugular catheter ; lymphangiograms obtained by direct injection of radio-opaque material through the jugular catheter, and a decrease in lymphocyte counts to 4% of the total white blood cell count, compared to 30% in the controls. There was no significant loss of weight in the surviving dogs.


[Granulocyte stem cells in a case of Chediak-Higashi-Steinbrinck syndrome].

Bone marrow from a child with Chediak-Higashi-Steinbrinck disease was cultured. It was found that all cells with granulocytic colony forming ability were affected by the disease. Cytoplasmic vacuoles were identified as lysosomes by their cytochemical staining. The cloning rates suggest that granulopoiesis is largely ineffective.


The effect of diuretic treatment on red cell and plasma sodium concentration in patients with hepatic cirrhosis and ascites.

The sodium content of red cells and plasma in some cirrhotic patients with ascites has been measured, before, during and after diuretic treatment. The results were compared with those found in 37 normal subjects and 42 untreated cirrhotic patients with ascites. In the 10 cirrhotic patients with ascites studied, the mean sodium content in erythrocytes before diuretic treatment was 9.95 (+1- SD 3.27) mEQ/kg, 27.15 (+/- SD 5.60) mEq/kg of dry weight and 15.02 (+/- SD 2.70) mEq/kg of red cell water. During diuretic treatment, the mean sodium content in red cells was significantly decreased to 6.27 (+/- SD 1.46) mEq/kg; 18.85 (+/- SD 4.36) mEq/kg of dry weight and 10.15 (+/- SD 2.41) mEq/kg of red blood cell water (P less than 0.01, P less than 0.01, and P less than 0.02 respectively). The mean plasma sodium concentration which was 135.95 (+/- SD 4.14) mEq/kg before diuretic treatment was significantly decreased during treatment to 129.19 (+/- SD 2.77) mEq/kg, P less than 0.001. Once the ascites had disappeared and the diuretic treatment had been discontinued the sodium concentration in both plasma and red cells became normal. The radioactive 22Na exchange "in vitro" between plasma and red cells of cirrhotic patients with ascites remained unmodified during diuretic treatment.


A model for the synthesis of glycoproteins.

A technique is described for assaying membrane bound enzymes. The results of studies with two sequential glycoprotein:glycosyl transferases are presented. The results are discussed in the light of recent suggestions concerning the structural organisation of these enzymes. A description, of the specificity of these enzymes for high molecular weight substrates, is presented.


Substrate inhibition kinetics in assemblages of cells.

The collective kinetic behavior of a linear array of cells containing a substrate inhibited enzyme is studied with a model in which each cell is considered a well-stirred compartment surrounded by a semipermeable membrane. At large values of a reaction-permeation modulus, wherein substrate access to interior cells is limited, plots of total reaction rate versus concentration of the external reservoir show sharp projections which correspond to dominant reaction occurring in a pair of symmetrically placed cells. Over prescribed ranges of reservoir concentration, multiple stable steady states can occur, some of which are characterized by asymmetric profiles of concentration and reaction rate across the array. A simple stability criterion is proposed and applied to arrays with arbitrary numbers of cells.


Dynamics of cellular growth.

The assumption that prompted the studies reported in this paper was that the unsatisfactory state of our knowledge on the regulation of cellular growth might derive from the reductionistic approach used to investigate it. Thus an analysis of cellular growth which applied concepts derived from systems dynamics was undertaken. First of all a dynamic model of cellular growth has been constructed. It has the following features: the levels of DNA, ribosomes and proteins are the defining levels; cellular growth is expressed by a close loop in which the level of ribosomes per genome and, indirectly, the level of proteins per genome are stabilized around goal values by the action of negative feed backs. The validity of the model has been tested by its ability to predict the growth kinetics of a real system (exponentially growing Neurospora cells). The simulated growth has been found to reproduce with great accuracy that of Neurospora cells. A slightly modified model, which takes into consideration also the degradation of ribosomes and of proteins, is shown to predict with accuracy the dynamics of growth of both growing and resting fibroblasts. These latter results suggest that the rates of macromolecular turnovers play a central role in the control of proliferation of mammalian cells: the condition of zero growth seems to be achieved when the rate of synthesis and the rate of degradation of proteins are the same. The possibility is discussed that the model indicates a unifying hypothesis of the mode of action of growth controlling conditions (hormones, growth factors, contact inhibition).


A computer simulation model for analysis of conformation of nuclear chromatin and of the transcription process.

Based on experimental data (see Lindigkeit et al., 1974) an algorithmic computer model was devised with the following parameters: (a) nbr. and position of hypothetic blocker sites at the DNA which inhibit transcription in such a manner that distinct RNA chain lengths arise; (2) time depending probability functions that a polymerase molecule (p.m.) is able to pass a blocker site; (3) time depending rate of viability of synthetizing p.m. and (4) distribution of the p.m. on the template at the time t0. For comparison between observed and computed results 3 parameters are used: (1) distribution of chain lengths of RNA molecules in vitro synthetized; (2) their total amount and (3) the number of active p.m. By means of comparisons between computed and experimental results it is possible to test hypotheses about internal structural and functional parameters of the system under investigation, e.g. estimation of the rel. influence of template vs. p.m. characters in the transcription process, hypotheses about the type of distribution of p.m. at time t0, their initiation and salt depending removal probability of the blocker structures.


Time hierarchy, equilibrium and non-equilibrium in metabolic systems.

A metabolic system consists of cooperating biochemical reactions. The motion is described by differential equations in the metabolites. The right-hand sides of these equations are linear combinations of the velocities of the individual reactions. These velocities depend in a non-linear manner on the metabolite concentrations (according to the law of mass action). A characteristic "metabolic" time may be defined for the motion of the whole system. It scales the essential metabolic events whose evolution time is comparable to this metabolite time unit. The constituent reactions of the metabolic system have an individual characteristic time which need not coincide with the general metabolic time. The individual time characterises the approach to the individual equilibrium of the isolated undisturbed reaction. According to the ratio of these two time scales, a single reaction may be fast, or slow, or essential, as compared with the metabolic events. Characteristic time of a single reaction and its steady-state deviation from equilibrium are closely related. It can be shown that the relative deviation from equilibrium of a reaction within the metabolic network is of the same numerical order as the ratio between individual time to metabolic time. The interaction of many reactions with different characteristic times introduces a time hierarchy into the system. This can be made transparent by appropriate scaling and by linear transformation of the system. The subsystem of fast cooperating reactions (dehydrogenases, phosphotransferases) attains a state which is near to the individual equilibrium and reestablishes this state after perturbation. The equilibration is fast; an ultrarapid phase of cofactor equilibrium can be distinguished from the fast phase of substrate equilibrium (exchange of metabolic material between different pathways). During the slower metabolic phase these near-equilibria manifest themselves as stoichiometric linkage between unrelated metabolites. The latter cease to be independent variables and combine to metabolic pools. It can be strictly shown that the essential variables at the metabolic time scale are carrier pools and the degree of occupancy of these carriers by metabolic groups. Chemically different types of carrier pools may be functionally linked together by fast reactions. A consequence of such an arrangement of reactions are distance effects: Changes at one end of a metabolic map may be directly conveyed to other pathways via stoichiometric linkage brought about by fast equilibration of cofactor reactions.


Analytic procedures for large dimention nonlinear biochemical oscillators.

An analytic method is presented which can be used to determine if the following system of nonlinear differential equations has periodic solutions x1 = h(xn)-b1x1 xj = gj-1xj-1-bjxj j = 2, ... n A systematic dual input describing function procedure is given for constructing a function of the reaction constants R, where if R greater than 1 a periodic solution exists and if R smaller than 1 there is no periodic solution. The form of R constructed generalizes immediately to an arbitrarily large dimension. The method generalizes to cover systems displaying hysteresis kinetics, systems subject to chemical noise, and systems containing delay components. The method has been applied to a well known biochemical problem where h(xn)-k/(1 + alphaxnrho). For rho = 1, for all n, there are no stable limit cycles such that xj(t) greater than O, t larger than or equal to O. For rho = 2,n larger than or equal to 8 it is possible to construct a parameter set such that stable oscillations appear.


Familial Prader-Willi syndrome.

Five cases of Prader-Willi syndrome were found in an inbred south Louisiana family. Pedigree data strongly support autosomal recessive inheritance of the syndrome in this family.