Isolation and properties of type II alveolar cells from rat lung. Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.


The failure of aerosolized superoxide dismutase to modify pulmonary oxygen toxicity. Superoxide (O2-.) is a highly toxic free radical that may be an important component of pulmonary O2 toxicity. The primary defense against this free radical is superoxide dismutase. Rats were exposed to aerosolized superoxide dismutase, and it failed to modify either the time course or the cumulative toxicity of 100 per cent O2. Because the aerosolized enzyme can be expected to be delivered only to the extracellular space of the lung, it is suggested that the primary site of production and of damage due to O2-induced free radicals must be within the intracellular space.


Lymphomatoid granulomatosis: association with retroperitoneal fibrosis and evidence of impaired cell-mediated immunity. Lymphomatoid granulomatosis is a non-neoplastic lymphoreticular disorder that usually presents as primary lung disease. A case of lymphomatoid granulomatosis associated with retroperitoneal fibrosis is reported. The retroperitoneal fibrous tissue contained the same variegated lymphoreticular infiltrate that formed the lung tumor, suggesting that the disease may have begun in the retroperitoneum. An extensive immunologic evaluation of the patient revealed impaired cell-mediated immunity.


Primary pulmonary sporotrichosis.

Primary pulmonary sporotrichosis. A 34-year-old alcoholic and drug addict developed cavitary pulmonary sporotrichosis that progressed slowly during 6 years. Pulmonary resection and pre- and postoperative therapy with amphotericin B were associated with prompt clinical improvement with no evidence of relapse during a 2-year follow-up. Histologic examination of lung revealed granulomatous inflammation with organisms consistent with Sporothrix schenckii, and interstitial talc (magnesium silicate) granulomas. The latter finding was consistent with the history of intravenous drug abuse. Although the presence of silicates in lung enhances the pathogenicity of some microorganisms, the relation of these findings to the pathogenesis of sporotrichosis in our patient is unclear.


Endobronchial foreign body demonstrated by xerotomography. A case of pneumonia secondary to aspirated foreign body is presented. Xerotomography was useful in detecting abnormalities of the tracheobronchial tree and in demonstrating endobronchial foreign bodies.


An urban epidemic of North American blastomycosis. Five culturally proved cases of blastomycosis occurred in a 9-month period during 1974 and 1975 in a western suburb of Chicago. All patients lived within a 1-mile radius of the center of Westmont, Ill. (population, 12,000). Three of these patients lived in adjacent buildings in the same apartment complex. None of the patients had significant outdoor exposure. Dust from a nearby construction site was postulated as a possible common source, although soil cultures from the area were negative. This is the third chronologic epidemic of the blastomycosis; a fourth one has been reported. It is the only outbreak to occur in a major metropolitan area.


Effects of beclomethasone dipropionate on tracheal mucous velocity. Beclomethasone dipropionate (200 micrograms, administered by inhalation to conscious sheep, does not affect tracheal mucous velocity during a 60-min period.


Respiratory abnormalities among grain handlers: a clinical, physiologic, and immunologic study. A survey of 300 grain elevator workers revealed that 77 per cent complained of eye symptoms; 64 per cent, of nasal symptoms; and 88 per cent, of one or more respiratory symptoms on exposure to airborne grain dust. Symptoms on exposure were independent of age and length of employment. Cough and wheezing on exposure were more common among smokers than nonsmokers (P less than 0.025). Nineteen per cent of the workers had had episodes of grain fever. The prevalence of chronic bronchitis was 37 per cent (42 per cent of smokers and 30 per cent of nonsmokers). Wheezes on auscultation were found in 23 per cent. Measurements of lung ventilatory function, as well as diffusing capacity, correlated significantly with age and smoking habits, but not with length of employment. Thirty-seven per cent of the workers had an abnormal mean forced expiratory flow during the middle half of the forced vital capacity (47 per cent of smokers and 13 per cent of nonsmokers), and 34 per cent had an abnormal maximal expiratory flow after exhalation of 50 per cent of the forced vital capacity (40 per cent of smokers and 13 per cent of nonsmokers), whereas only 13 per cent had an abnormal ratio of 1-sec forced expiratory volume to forced vital capacity. There was no correlation between precipitins to fungi, bacteria, grain, or grain dust antigens and acute or chronic respiratory symptoms, lung function, or grain fever. There was, however, a significant correlation between cutaneous reactivity to grain dust and wheezing on exposure (P less than 0.02). Abnormal flows at low lung volumes were more common among cutaneous reactors to common allergens. We concluded that exposure to airborne grain dust can cause acute inflammatory reaction to the exposed mucosa, and it is highly probable that grain dust contributes and, in some cases, causes chronic airway disease.


Sequential immunologic stimuli to the respiratory tract: a paradox in the degree of potentiation of airway responses depending on the sequence of reverse passive and active immunologic stimulation. Immunologic stimuli to the airway of rhesus monkeys were given by aerosol challenge with ascaris antigen or anti-IgE. Both of these stimuli produce immediate-type airway responses. When 2 sequential aerosol challenges were given during the same experiment, the response following the second stimulus was always less than or equal to the first response following any combination of stimuli except the anti-IgE-ascaris sequence. The second response following the latter challenge was always greater than or equal to the first response. The possibility that this exception was the result of anti-IgE priming mediator releasing cells for antigen was not supported by in vitro experiments. It is suggested that the results obtained with the anti-IgE-ascaris sequence may relate to the presence of intralumenal mast cells in the bronchi and the molecular weights of the 2 immunologic stimuli.


Airway response to carbachol in normal and asthmatic subjects: distinction between bronchial sensitivity and reactivity. By constructing cumulative dose-response curves to inhaled carbachol in 12 normal and 17 asthmatic subjects with comparable baseline specific airway conductance, we have shown that there were wide variations among subjects in the dose of carbachol needed to cause a 25 per cent decrease in specific airway conductance (bronchial sensitivity) and in the slopes of the curves (bronchial reactivity). Furthermore, there was no significant correlation between these 2 characteristics of the bronchial response to carbachol. The mean dose-response curves of the asthmatic and the normal subjects were widely divergent, indicating that the asthmatic subjects differed from normal subjects more in terms of bronchial reactivity than in bronchial sensitivity. This suggests that different mechanisms determine the sensitivity and reactivity of the bronchial tree, and that hyper-reactivity is the main feature of the asthmatic response. Both should be assessed when the bronchial response to bronchoconstrictor agents is measured.


Theophylline bioavailability: a comparison of the oral absorption of a theophylline elixir and two combination theophylline tablets to intravenous aminophylline. The bioavailability of theophylline from single doses of an elixir (Elixophyllin) and two different tablet formulations, as compared to intravenous aminophylline, was studied with a crossover design in 12 normal volunteers. Both tablet formulations (Marax and Tedral) contain ephedrine. Marax contains hydroxyzine hydrochloride, and Tedral contains phenobarbital. The absorption of theophylline was most rapid from the elixir, whereas that from Marax was faster than that from Tedral. The peak concentrations of theophylline after administration of the 3 oral dosage forms were in the order, elixir greater than Marax greater than Tedral, however, the time to achieve peak concentration was highly variable and did not differ significantly among the 3 products. On the basis of area under the serum concentration-time curves, the absorption of theophylline from the elixir and from Marax was essentially complete. The area under the serum concentration curve after administration of Tedral was significantly less than that after intravenous aminophylline, elixir, and Marax; however, when the individual areas under the concentration curves were adjusted for intrasubject variation in elimination rate constant, the mean area under the concentration curve after Tedral no longer differed significantly from those of the intravenous and other two oral products. A large degree of intersubject variation in the oral absorption of theophylline was observe din this study. Therefore, in addition to the well-documented, large individual variation in the serum clearance of theophylline, intersubject differences in the absorption of the drug is another factor that complicates proper adjustment of the dose in oral theophylline therapy.


An animal model of cigarette smoking in beagle dogs: correlative evaluation of effects on pulmonary function, defense, and morphology. Using beagle dogs, we have developed an animal model for the evaluation of the effect of chronic cigarette smoking on pulmonary defense and function and on lung structure. The smoking apparatus developed allows the dog actively to inhale properly diluted (1:4) smoke directly from the cigarette through a mouthpipe. In this model, 6-month and 1-year periods of mild to moderate smoking caused impairment of tracheal mucociliary transport and bacteriosuppressive activity of alveolar macrophages, with little change in pulmonary function. Morphologically, subtle, but significant, lesions were noted in the central airways and bronchiolar walls, consisting of tracheal epithelial basal cell hyperplasia, proliferation of goblet cells in central airways, and peribronchiolar infiltration by inflammatory cells. Morphometry of bronchiolar size distribution, volume proportion of parenchymal structure, and alveolar surface area, however, failed to show significant differences between the groups.


A comparative study of the mechanical properties in aging alveolar wall. Alveolar wall from the lung of aging humans shows a progressive decrease in maximal extensibility, which should follow an increase in resting tissue length rather than a reduction in maximal length. An increase in resting tissue length is compatible with the change in lung volumes and reduction in elastic recoil that occurs with time. A model of the lung was used to compare the effects of a change in resting tissue length in diminishing elastic recoil with that of a reduction in the volume density of the elastic elements (emphysema). Such differentiation is important in selecting an animal that may model the aging or emphysematous lung. In the rat, rabbit, and horse, alveolar walls show no decrease in maximal extensibility with age. In the male monkey (M. nemestrina and M. mulatta) between birth and 2.4 years there is a decrease in maximal extensibility that lacks significance for the limited age span examined. On the other hand, the energy loss in length-tension cycling (hysteresis) of alveolar wall increases in aging humans, diminishes in rats and rabbits, and shows little change in horses and monkeys. The breaking force of alveolar wall increases with age in rats and rabbits but does not change significantly in the other species. Of these species, the monkey promises a better model of the age-related changes in maximal extensibility of alveolar wall. A measure of maximal extensibility can distinguish the effects of dilatation of air spaces from those of destruction of alveolar wall in causing loss of lung elastic recoil.


Pulmonary hyalinizing granuloma.

Pulmonary hyalinizing granuloma. Twenty patients with pulmonary nodules consisting of concentric hyaline lamellae, usually accompanied by perivascular collections of plasma cells and lymphocytes, were studied. In most instances, the lesions were multiple, bilateral, and mildly symptomatic. Many of these nodules showed all of the staining characteristics of amyloid, but others had an atypical birefringence pattern. No infectious agents were identified, and no consistent pattern of dysproteinemia was observed. Two of the patients had prior histories of tuberculosis. In other cases, the nodules were of unknown origin and pathogenesis. Four cases were complicated by sclerosing mediastinitis, and one, by retroperitoneal fibrosis and amyloidosis. Our current working hypothesis is that these lesions represent an exaggerated and, possibly, continuing immune response, perhaps to one of a number of agents.


[Regulation of the breadth of the field of self recognition]. On the basis of certain cybernetic considerations, the need is demonstrated of several plasmatic circuits controlling the amplitude of the self recognition field. Regulating every individual circuit in a defense-enhancing direction is demonstrated to trigger a self-destroying, increasing-positive-retroaction mechanism, which is in turn controlled by an increasing-negative-retroactive defense-inhibiting mechanism. The existence is also demonstrated of a correlation between these retroaction processes. When certain stimulation levels are exceeded, such a correlation results in the growth of the negative process being sped up. The possibilities are discussed of stimulating the regulation of the circuit that governs the tumoral defense, keeping the negative retroaction below the limits at which it starts developing exponentially, thereby enhancing anticancer defenses.


Evidence for regression of advanced atherosclerotic plaques. The purpose of this presentation is to summarize briefly the evidence, much of it recent and direct, that the most prominent lesion components of the advanced atherosclerotic plaques in experimental animals, namely the necrotic center and the intracellular lipid, can undergo substantial regression. Some of the data will be reviewed indicating that this improvement of lesions in size, consistency and safety is due to a combination of remodeling, regression and healing. The recent evidence will be included indicating that human lesions can also respond favorably to the same rigorous serum cholesterol lowering regimens that lead to improvement of the advanced monkey and swine plaques.


The emerging cellular pathobiology of atherosclerosis. The purpose of this paper is to present a brief coordinated overview of the recent results of research at the artery cell level which appear to have the greatest impact on the rapidly improving understanding of the pathogenesis of atherosclerosis in humans. The majority of these studies have employed in vitro methods and utilized the tools of modern cellular and molecular biology. These include microdissection; cell separation; tissue or cell culture; enzyme, lipid and protein chemistry as well as immunochemistry, ultrastructural visualization, cell organelle and membrane fractionation and the use of genetic markers. With these tools it is possible to study the interaction of the major space-occupying cells of the atherosclerotic plaque (especially the arterial smooth muscle cells) with many of the blood components, especially the lipoproteins and the other serum factors that appear to influence cell division. This direction of study appears to usher in a new era of atherosclerosis research.


[Evaluation of certain hemostasis parameters in premature infants]. In order to evaluate the specificity of hemostatic mechanism in premature Infants, the following examinations of coagulation and fibrinolytic parameters have been done: platelet number, concentrations of fibrinogen prothrombin, factors V, VII X, X, VIII, IX and XIII, antithrombin III, plasminogen, euglobulin lysis time, fibrin degradation products, alpha-1 antiplasmin and alpha-2 macroglobulin. The examinations have been done in the whole of 18 premature infants of both sexes. The obtained results show that lowered level of coagulant activity was not exclusively the consequence of low activity of vitamin-K dependent factors, but the result of more complex disorders partly connected with the transient reduction of factors VIII, XIII and fibrinogen. The total fibrinolytic activity, in the meantime, was of the normal intensivity. The established disorder of coagulation-fibrinolytic balance probably represents the certain contributing factor in more frequent occurrence of haemorrhagic syndrome in premature infants.


[Isoelectric spectrum of IgD in myeloma]. Isolated and highly purified myeloma IgD DEK, STA and SAR were subjected to isoelectric focusing in thin layer of polyacrylamide gel using equipment and PAG plate 3, 5-10 from LKB. Although homogeneous in electrophoresis on cellulose acetate folien, immunoelectrophoresis and DISC PAA gel electrophoresis analysed IgD showed high isoelectric heterogeneity. They formed isoelectric spectra with 24-27 pl zonnes ower the pH range 5,4-9,3. Based on densitometric analysis of gel stripps zonnes with a small protein content were excluded from calculation of the real isoelectrical range. According to that manipulation isoelectrical range was determined as pH 6-8. Heterogeneity of isolated myeloma IgD may be due to the post-synthetic transformation of molecules in vivo as well to degradation and/or aggregation of IgD in vitro during the preparation of samples for isoelectrofocusing. However, myeloma IgD are in fact more heterogeneous in isoelectrofocusing than myeloma immunoglobulins of other classes.


[Case report of congenital deficiency of factor V and factor VIII]. A case of a rare congenital combined deficiency of factor V (14%) and factor VIII (30%) is described for the first time in our literature. Nine years old girl was admitted to hospital because of nightly bleedings from hyperplastic and inflamed gums. Clinical picture was mild what is in accordance with the results of coagulation studies. Under substitution therapy tooth extraction passed without complications.


[Albumin and its therapeutic use - part I]. Albumin is most abundant and most studied protein of the circulation. Its biosynthesis is closely dependent on the nutrition, amino acid supply, hormonal millieu, environment, osmotic equilibrium, diseases and some other factors. Albumin is synthetized in the liver on a polyscmes and delivered into the blood streem. Degradation of albumin is practically still unknown and is one of many biological puzzles. Albumin prepared for therapeutic use almost contains dimers, oligomers and polymers which are very important, because they are one of the parameters for evaluation of albumin products quality.


[Albumin and its therapeutic use - Part II]. Albumin is the most effective oncotic agent and has wide clinical application. In the resuscitation of injured patients with hypovolemic shock (during war and peace) the use of supplemental albumin is purported to be more effective in restoring plasma volume, increasing cardiac output, preventing pulmonary oedema and maintaining organ perfusion than resuscitation without supplemental albumin. Most important in a study of albumin are criterias for appropriate use because it was sometimes unjustified. Therapeutic use of albumin should not be dictated by arbitrary or pragmatic restriction but rather by rational prescribing from physicians well educated in its use.


[Haptoglobin levels in premature infants]. A group of 30 preterm babies with no evidence of infections was investigated. They were separated in 3 groups according to the gestational age. In the first group, which included babies 28 to 32 weeks of gestational age, haptoglobin was detected in 5, but in 3, levels were under 25 mg/dl. In the second group, which consisted of babies of 33 to 36 weeks of gestational age, haptoglobin was detected in 6 infants, with levels less than 25 mg/dl in 3. Finally, in the group of babies of 37 to 40 weeks, haptoglobin was presented in 7. Haptoglobin was detected in 18 infants, which means in 60% of patients. Levels of haptoglobin were lower in babies of lower gestational age. Values obtained for haptoglobin levels in this study will be used in diagnostic purposes, for comparation with levels obtained in various pathological conditions.


Activities of enzymes in plasma should be measured at 37 degrees C. There are considerable differences of opinion as to the most desirable temperature at which activities of enzymes in plasma should be measured. Debate centers around 25 degrees C because of its widespread use in physico-chemical measurements, 30 degrees C because of its supposed technical convenience, and 37 degrees because of its physiological significance. There is, however, widespread agreement that the activities of intracellular enzymes, and of enzymes having a natural function in plasma, be measured at 37 degrees C. Perhaps because estimations of the activities of those enzymes in plasma that have leaked from damaged tissues have been subject to almost continuous modifications, temperature has simply become just another variable to take into account. In this article I describe the relevance of a temperature near 37 degrees C to the activities of all enzymes in man, intracellular and extracellular. I conclude that 37 degrees C is to be favored as the temperature for assaying all parameters in human biochemistry affected by temperature. These include not only enzyme activities but also pH, blood-gas equilibria, binding data, and, in fact, all kinetic activities in the body.


Secondary hypertriglyceridemia and hyperlipoproteinemia in patients with primary asymptomatic gout. We carefully selected 30 men with primary gout, rendered asymptomatic by therapy, to examine the frequency and type of hyperlipidemia and hyperlipoproteinemia, with the objective of determining whether serum uric acid, alcohol intake, liver function, kidney function, and (or) drugs were participating in the secondary lipid disorder. Sixty-one age- and sex-matched men were used as controls. About 73% of the gout patients had hypertriglyceridemia, 1.6-fold the frequency found in the control group. Types IV and IIb lipoprotein electrophoretic patterns were most prevalent in the gout group. Neither alcohol intake nor hyperuricemia, per se, seems to be the cause of the lipid and lipoprotein disorder and cannot be related to liver or kidney dysfunctions. Obesity was the major underlying factor associated with the lipidemia. The study suggests that diet and, possibly, defective clearance of triglycerides may be etiologic factors associated with the abnormal serum triacylglycerol (triglyceride) and lipoprotein concentrations in these individuals.


Comparison of interlaboratory results for blood lead with results from a definitive method. Results reported by 113 participants in the Blood Lead Proficiency Testing Program conducted by the Center for Disease Control were compared with those obtained by the National Bureau of Standards (NBS) with a definitive methods (mass spectroscopy-isotopic dilution) for blood lead analyses. Data were compiled from the results obtained for 12 whole-blood samples containing 1.5 g of disodium EDTA per liter. Twelve separate blood samples were obtained from cattle which had been given lead nitrate orally. Lead concentrations in the samples ranged from 0.628 to 4.93 mumol/L (130-1020 micrograms/L) as determined by NBS. The methods used by laboratories were classified according to six basic groups: anodic stripping voltametry; and atomic absorption spectroscopy in which either extraction, carbon rod, graphite furnace, tantalum strip, or Delves cup was used. For results obtained in each group a linear regression analyses of laboratory values was made on the basis of NBS values. In comparison to the definitive method, most field methods for blood lead tended to overestimate the lead concentration when the actual lead concentration was less than 1.96 mumol/L (400 micrograms/L) and to underestimate the lead concentration when the actual lead concentration was greater than 2.45 mumol/L (500 micrograms/L).


Influence of a between-run component of variation, choice of control limits, and shape of error distribution on the performance characteristics of rules for internal quality control. A computer-stimulation study has been performed to determine how the performance characteristics of quality-control rules are affected by the presence of a between-run component of variation, the choice of control limits (calculated from within-run vs. total standard deviations), and the shape of the error distribution. When a between-run standard deviation (Sb) exists and control limits are calculated from the total standard deviation (St, which includes Sb as well as the within-run standard deviation, Sw), there is generally a loss in ability to detect analytical disturbances or errors. With control limits calculated from Sw, there is generally an increase in the level of false rejections. The presence of non-gaussian error distribution appears to have considerably less effect. It can be recommended that random error be controlled by use of a chi-square or range-control rule, with control limits calculated from Sw. Optimal control of systematic errors is difficult when Sb exists. An effort should be made to reduce Sb, and this will lead to increased ability to detect analytical errors. When Sb is tolerated or accepted as part of the baseline state of operation for the analytical method, then further increases in the number of control observations will be necessary to achieve a given probability for error detection.


Liquid-chromatographic analysis for methylphenidate (Ritalin) in serum. We describe a "high performance" liquid chromatographic method for quantitating methylphenidate in serum. The internal standard, 4,5-diphenylimidazole, and  serum or plasma sample are extracted in chloroform, evaporated, and redissolved in 20 mmol/L potassium phosphate (pH 3.5)/high-purity acetonitrile, 80/20 by vol. A centrifuged aliquot is chromatographed on mu-Bondapak C-18 with the phosphate/acetonitrile solvent as mobile phase, a flow rate of 1.6 mL/min, and a column temperature of 40 degrees C. Absorbances are read at 192 nm. This method reliably measures concentrations greater than 20 micrograms/L and has analytical recoveries of 74%.


Liquid chromatographic analysis of disopyramide and its mono-N-dealkylated metabolite. We describe a rapid, sensitive, and specific "high performance" liquid chromatographic analysis for disopyramide and its mono-N-dealkylated metabolite in serum, urine, and saliva. We used a mu-Bondapak CN column and an acetate buffer mobile phase containing methanol. Retention times for the two compounds and the internal standard, p-chlorodisopyramide, were 3.4, 4.1, and 6.3 min, respectively. The lower limits of sensitivity for drug and metabolite were 50 and 80 micrograms/L, respectively, with maximum coefficients of variation of 4.6 and 12%, respectively. Currently used antiarrhythmic drugs did not interfere with the analysis of disopyramide, and the pharmacokinetics of the drug, obtained from studies of one subject, agree well with reported values.


Liquid-chromatographic determination of acetaminophen in serum. We describe a sensitive and precise "high-pressure" liquid-chromatographic method for determining acetaminophen in serum. The 2-acetaminophenol and 3-acetaminophenol structural isomers of acetaminophen are used as internal standards. The method, which involves solvent extraction and adsorption chromatography on silica, provides excellent sensitivity, accuracy, and selectivity. The standard curve is linear over the range of acetaminophen concentrations of 0.5 to 300 mg/L, which makes the method useful for both pharmacokinetic studies and overdose monitoring. Analytical recovery is 97% for acetaminophen concentrations ranging from 5 to 300 mg/L. Many commonly used drugs were tested and found not to interfere. The procedure has been successfully adapted as a microscale method requiring only 50 microL of sample. The microscale method is particularly useful for pediatric and neonatal patients for whom sample size is a major concern.


An evaluation of ethylene glycol-based liquid specimens for use in quality control. We investigated the analytical acceptability of ethylene glycol-based control sera by preparing sets of aqueous and ethylene glycol-based specimens that had added uric acid, calcium, creatinine, glucose, urea, sodium, and potassium. Ethylene glycol caused a significant systematic proportional bias in procedures involving dialysis, but had no discernible effect on methods not involving dialysis. The extent of bias was proportional to the concentration of ethylene glycol, was independent of protein concentration, and differed according to the type of dialysis system used. We conclude that ethylene glycol-based control materials can have useful applications in clinical chemistry, but caution must be exercised in their use with analytical systems that employ dialysis.


Cytosine and orotic acid in urine of immunodeficient children. We describe procedures for determining cytosine and orotic acid in urine. We determine cytosine by cation-exchange analysis with either HCl or pH 5.2 buffer as eluent. Orotic acid is first separated by an anion-exchange separative procedure; after lyophilization, the product is subjected to "high-pressure" liquid chromatography for further separation and detection. We analyzed urine from normal subjects and from immunodeficient children. Three children with severe combined immunodeficiency had increased levels of cytosine in urine (23-160 mmol/mol creatinine); one child with severe combined immunodeficiency and two children with other immunodeficiencies had normal urinary levels (less than 2 mmol/mol creatinine). Orotic acid excretion in urine was normal (1-5 mmol/mol creatinine) in all of th immunodeficient children. We discuss the possible significance of the increased cytosine excretion in the three children with severe combined immunodeficiency.


Measurement of alpha-, beta-, and gamma tocopherol in serum by liquid chromatography. A liquid-chromatographic assay for alpha, beta, and gamma-isomeric tocopherols in human serum is reported. The tocopherols and the internal standard (tocol) are absorbed into a silica gel column and are eluted in less than 10 min with n-hexane/isopropanol (99.4/0.6, by vol) at a flow rate of 1 mL/min. The complete analysis requires no longer than 30 min. Within-day precision (CV) was 1.4% (mean = 13.18 mg/L, n = 24), 7.4% (mean = 0.214 mg/L, n = 14), and 1.3% (mean = 1.01 mg/L, n = 14) for alpha-, beta-, and gamma-tocopherol, respectively. Day-to-day precision (CV) was 4.4% (mean = 9.85 mg/L, n = 10) for alpha-tocopherol, 9.1% (mean = 0.222 mg/L, n = 20) for beta-tocopherol, and 3.8% (mean = 1.00 mg/L, n = 20) for gamma-tocopherol. Extraction recoveries for alpha-, beta-, and gamma-tocopherol averaged 92.4 +/- 2.9% (n = 5), 91.4 4/- 7.6% (n = 5), an 92.0 +/- 4.1% (n = 4), respectively. The smallest injected amount detectable is estimated to be 0.03 micrograms for alpha-, and 0.04 micrograms for beta- and gamma-tocopherol.


Effects of wavelength error and spectral band width on measurement of alkaline phosphatase activity. We re-examined the effects of wavelength error and spectral band width on the measurement of alkaline phosphatase activity. The methods we studied is relatively insensitive to these two factors, a conclusion we base on both experimental results and theoretical analysis. These findings are in conflict with a recently published report [Lott et al., Clin. Chem. 24, 938 (1978)], and we suggest a possible explanation for this.


Incorrect least-squares regression coefficients in method-comparison analysis. The least-squares method is frequently used to calculate the slope and intercept of the best line through a set of data points. However, least-squares regression slopes and intercepts may be incorrect if the underlying assumptions of the least-squares model are not met. Two factors in particular that may result in incorrect least-squares regression coefficients are: (a) imprecision in the measurement of the independent (x-axis) variable and (b) inclusion of outliers in the data analysis. We compared the methods of Deming, Mandel, and Bartlett in estimating the known slope of a regression line when the independent variable is measured with imprecision, and found the method of Deming to be the most useful. Significant error in the least-squares slope estimation occurs when the ratio of the standard deviation of measurement of a single x value to the standard deviation of the x-data set exceeds 0.2. Errors in the least-squares coefficients attributable to outliers can be avoided by eliminating data points whose vertical distance from the regression line exceed four times the standard error the estimate.


Automated measurement of total cholesterol and triglycerides, in "tandem," on the discrete sample analyzer, Gilford System 3500. We have developed an automated procedure on a discrete sample analyzer, Gilford System 3500, which alternately measures both total serum cholesterol and triglyceride concentrations as a "tandem" procedure. We used Dow Diagnostic's fully enzymatic, colorimetric reagents and aqueous standards to calculate unknowns ratiometrically. Cholesterol and then triglyceride reagent are dispensed into alternate cups; the produced color at 500 nm is measured after an ambient temperature incubation of 20 min. Reagent and sample carryover is less than 1.6%. Correlation coefficients of 0.997 for comparison for both automated tests with the manual methods at 30 degrees C and a typical CV of less than 2.0% show this "tandem" procedure to be reliable and accurate.


Determination of catecholamines and their 3-O-methylated metabolites in urine by mass fragmentography with use of deuterated internal standards. We report the determination of catecholamines (dopamine, norepinephrine, and epinephrine) and their 3-O-methylated metabolites (3-methoxytyramine, normetanephrine, and metanephrine) in urine by gas chromatography/mass spectrometry, with use of stable isotopically labeled internal standards. Normal excretion values in terms of creatinine, expressed as a function of age, are given, together with results obtained for patients with neuroblastoma, ganglioneuroblastoma, pheochromocytoma, parkinsonism treated with L-DOPA plus peripheral decarboxylase inhibitor, and a patient with cardiogenic shock treated with dopamine. The results obtained for normal adults are discussed in relation to other catecholamine metabolites and mean overall catecholamine turnover.


Macro creatine kinase BB in serum, and some data on its prevalence. We report the case of a patient with persistently above-normal activity of creatine kinase (CK) in serum, a major fraction of which on electrophoresis moved as a band between the MM and MB isoenzymes and on anion-exchange column chromatography eluted in the MB fraction. Measurements in the presence of specific M or B subunit-inhibitory antibodies indicated that 93% of the activity consisted of B-isomers. From these experiments we conclude that the abnormal CK is of BB nature. Gel filtration and immunoglobulin precipitation showed that the CK-BB was complexed with IgG. Normal CK-BB, when mixed with the patient's serum, was converted to macro CK-BB. In vitro stability of 37 degrees C of the abnormal enzyme was much greater than that of normal BB and MM isoenzymes. Following this finding, we then assessed 310 sera, received for enzyme assay by the clinical laboratory, for electrophoretically abnormally migrating CK isoenzymes. Of these, five (1.6%) contained such enzymes, all being of BB nature. They were of increased molecular mass, and at least three of them were complexed with IgG.


Manual fluorometry of phenylalanine from blood specimens collected on filter paper: a modified procedure. In this manual fluorometric method, blood samples are used that have been impregnated on the filter paper, a convenient collection technique that is widely used to screen newborns for phenylketonuria. The modified procedure, based on the method of McCaman and Robins [J. Lab Clin. Med. 59, 885 (1962)], includes elution of phenylalanine from specimens on filter paper, removal of proteins by precipitation with trichloroacetic acid at 0 degrees C, and then reaction with ninhydrin-peptide reagent for color development. The standard curve is linear to at least 200 mg/L and the CV is 6.3% for a phenylalanine concentration of 27 mg/L. The modified procedure is suitable both for early screening for phenylketonuria and for monitoring blood phenylalanine of phenylketonurics during dietary therapy.


Spectral analysis of urinary reactions: a preliminary study based on a novel concept. The concept of spectral analysis of urinary reactions (a range of simple chemical tests combined with pattern analysis) is described. A preliminary study in which we used 10 nonspecific tested on untimed urine specimens from 20 controls and 20 diabetics, matched for age and sex, suggested that a combination of these tests could be used to separate diabetics from healthy subjects. This combination, however, did not prove satisfactory for a further group of 13 diabetics. Closer examination revealed that factors such as therapy and glycosuria affected the results of these tests. This approach, which should extend the usefulness of urinalysis, may prove viable when larger groups, and possibly a different set of tests, are used to establish a more robust combination of tests.


Improved lysozyme assay in biological fluids. We describe a simple, rapid, sensitive, and highly reproducible assay for lysozyme, with use of concentrated cell suspensions of Micrococcus lysodeikticus in Tris-buffered glycerol/water (40/60 by vol), pH 7.5. Stored at -20 degrees C, the cells' susceptibility to lysozyme remains unaltered over long periods. Almost identical concentration curves were obtained with different aliquots of the same preparation during eight months. Lysozyme activity was reflected in the decrease in absorbance of the reaction mixture after incubation for 15 min at 37 degrees C. Concentrations of egg-white lysozyme as low as 0.02 mg/L can be accurately assayed.


Clinical evaluation of thyroid CT values in various thyroid conditions. CT scans of the thyroid were performed with a Hitachi CT-W2 device. The CT value of the region of interest (ROI) was obtained from CT images of the thyroid. Mean and standard deviation was 112 +/- 10 in 44 normals. Out of 27 cases of hyperthyroidism, the CT value of 13 untreated cases of 58 +/- 4, and that of 13 cases under treatment was 73 +/- 7 in average. The CT value of the single cured case was 100. Twelve cases of chronic thyroiditis and 5 cases of hypothyroidism showed averages of 57 +/- 11 and 36 +/- 13, respectively. The results of the above-mentioned clinical study indicate the estimation of the CT value of the thyroid is useful for diagnosis of various thyroid conditions.


CT anatomy of the nasopharynx, nasal cavity, paranasal sinuses, and infratemporal fossa. Analysis of anatomical features of the nasopharynx, nasal cavity, paranasal sinuses, and infratemporal fossa was carried out by retrospective review fo 50 CT scans. Conventional and axial anatomy are discussed, and normal size limits of selected structures are proposed on the basis of a series of measurements. Examples of early and advanced disease are shown.


Computed tomography of intracerebral gangliogliomas. CT and clinical findings in 6 histologically proven cases of intracerebral gangliogliomas (out of 14,000 cranial CT examinations) are presented. The tumor is most often isodense, with areas of calcification. Even though CT findings are nonspecific, they are suggestive of the benign nature of the tumor. When this is histologically confirmed (by biopsy) the more aggressive forms of treatment may be obviated.


Renal arteriovenous malformation presenting as a mass lesion. Arteriovenous malformations of the kidney are definitively diagnosed by selective arteriography (1). The following case is unique in that it presented as an avascular mass and the lesion itself could not be demonstrated angiographically.


Evaluation of subarachnoid hemorrhage and cerebral vasospasm by computed tomography. The CT findings in patients with subarachnoid hemorrhage include visualization of the aneurysm (10% of the cases), subarachnoid, intraventricular, or intracerebral hemorrhage (87% of cases scanned within five days), "ischemic" lucency secondary to spasm (10%), and hydrocephalus (54%).


Computed tomography of choroid plexus lesions. Computed tomographic (CT) and clinical findings in 14 patients with 17 lesions of the choroid plexus are presented. Primary neoplasms are the most common lesions in this series and include papillomas, meningiomas, carcinoma, astrocytoma, and teratocarcinoma. There are five vascular lesions, consisting of cavernous angiomas, hemangiomas, and an arteriovenous malformation. One patient had bilateral xanthogranulomas. There was association between cavernous angiomas and Sturge-Weber, and between meningiomas and neurofibromatosis. Useful distinguishing clinical and CT features between the different types of lesions are discussed.


Mass lesions of the retroperitoneum (excluding specific organs). The value of computed tomography (CT) in the diagnosis and management of retroperitoneal masses is discussed. Neoplastic, inflammatory, and hemorrhagic conditions are shown. In addition to facilitating the detection of these mass lesions, CT is especially valuable in allowing accurate biopsies and aspiration to be performed and in the follow-up of the patient.


Normal anatomy and limitations in CT interpretation of lymph node disease. The CT appearance of normal retroperitoneal lymph nodes has been described. In many instances the structures are too small to be identified. other retroperitoneal structures, such as collapsed bowel loops, vessels, and other perirenal structures, may simulate the presence of nodes. CT is of great benefit in disease with bulky tumors, such as non-Hodgkin's lymphoma, testicular tumors, etc. Its usefulness is much more limited in disease that may have extensive nodal involvement but no significant enlargement of the nodes. The accuracy of CT scanning in Hodgkin's disease and in many instances of genitourinary tumors is questioned, and we submit that further studies are needed to establish the reliability of this mode of examination.


Subdural hygroma: diagnosis with computed tomography. Nine cases are reported that fulfilled the criteria for diagnosis of subdural hygroma. With the availability of serial computed tomographic (CT) scans, it is possible to document non-invasively the appearance of CSF-density subdural fluid where no prior blood accumulation has been present.


Portal vein ectasia simulating a vascular lesion in the pancreatic head in an enhanced CT scan. An ectatic portal vein in a patient with portal hypertension and chronic active hepatitis simulated a vascular lesion in the head of the pancreas in a contrast-enhanced CT scan.


The role of radionuclide brain imaging and computerized tomography in the early diagnosis of herpes simplex encephalitis. We have reviewed the medical records and radiographic examinations of 12 patients with herpes simplex encephalitis to assess the role of RN and CT in the early diagnosis of this disease. The initial RN study was positive in 83% (10/12) of cases while the initial CT study was positive in 75% (9/12) of cases. The earliest positive RN was seen on the second day after the onset of neurologic signs or symptoms while the earliest positive CT was seen on the third day. We describe various abnormal patterns encountered in HSE and discuss their diagnostic reliability. We make recommendations for the diagnosis of HSE based on our findings and on the information available in the literature


Tonsillectomy in Manitoba: who are the patients? the surgeons? the hospitals? Although tonsillectomy-adenoidectomy rates are declining across North America, this paper suggests they are not falling fast enough. More than half the cases coming to surgery fail to meet accepted standards regardless of who performs the surgery, or where it takes place. Family surgery is still fairly common and is almost never indicated. While younger physicians have higher standards of selection, they are not doing any less T and A surgery than their older colleagues; in fact younger general surgeons are doing more. This study concludes that non-indicated T and A surgery is still a prevalent problem, deserving of widespread attention.


Where do cancer patients die? A review of cancer deaths in Cuyahoga County, Ohio, 1957--1974. The great concern over the status and care of the dying cancer patient requires the understanding of current trends in care. An 18-year review of 55,288 death certificates of patients with cancer in Cuyahoga County, Ohio (1957--1974) revealed that 35,381 patients (65%) died in acute and chronic care hospitals, 8,251 patients (15%) died in nursing homes, and 11,242 patients (20%) died at home. Trends over the 18-year period demonstrated a shift from patients dying at home to patients dying in nursing homes. The hospital care of dying cancer patients remained unchanged during the study period. An analysis of 33 consecutive patients dying of cancer over a six-month period in an acute care hospital in Cuyahoga County showed an average length of stay of 20.1 +/- 15.7 days, during which only palliative care was provided. The cost benefit of home care/hospice programs is related to the final hospital stay of the dying cancer patient.


Longitudinal study of career choices of a SUNY-Upstate cohort of medical students. A SUNY-Upstate cohort was observed during the four-year matriculation (1974--1978) of the students for changes in their career choices. Medical school exposure seemed to increase students' interest in all major specialties, with the exception of family practice for which there was an overall 3% decrease. Internship and residency appointments accepted indicated that 75% entered primary care specialties, 8% more than the national average for 1978.


Recent status of detection, treatment, and control of hypertension in the community. The Hypertension Detection and Follow-up Program (HDFP), a national collaborative study, screened approximately 159,000 people for high blood pressure in 14 communities between 1973 and 1974. Results show that detection, treatment, and control of high blood pressure has improved considerably since the 1960s. Whereas in the past about half of all hypertensives knew they had high blood pressure, half of those detected were under treatment, and half of those under treatment had their high blood pressure under control, the corresponding percentages in the 14 HDFP communities a decade later indicate that 75% of hypertensives were detected, 72% of those were under treatment, and 70% of those under treatment had a diastolic blood pressure under 95 mm Hg. While the differences in prevalence of hypertension are between races rather than sexes (with black individuals in some age groups being about twice as likely as white individuals to have hypertension), the differences in detection and treatment rate are largely between sexes and not between races. Women are considerable more likely to be aware of their hypertension, to be under treatment for it, and to have their high blood pressure under control. Rates of control vary considerably among age-sex-race subgroups, from only 8% of white male hypertensives aged 30-39 to 67% of white female hypertensives aged 60--69. It appears that although efforts to combat this disease over the past decade have probably made considerable progress in improving the recognition and treatment of high blood pressure, there remain a large number of undetected, untreated, and uncontrolled hypertensives.


Determinants of dropout rate among hypertensive patients in an urban clinic. Noncompliance with follow-up is a serious problem in the management of hypertension. A retrospective cohort study examined dropout rates and their determinants among 249 randomly selected outpatients with essential hypertension from the medical clinic of an urban teaching hospital. Data were abstracted from hospital records and a subset of dropouts was interviewed. A lifetable analysis revealed that patients who were initiating therapy or who had been under therapy for less than six months had a 50% chance of remaining in care two years later, while 70% of patients who had been under therapy for more than six months at entry were still in care after this period. Patients who were less severely ill by several indicators were the most likely to drop out. It is hypothesized that the low perceived severity of illness, coupled with the costs and inconvenience of care and the lack of physician enthusiasm for the treatment of mild hypertension leads to non-compliance with follow-up.


[Complete testicular feminization syndrome and poorly-differentiated lymphocytic lymphoma. Report of a case]. A case of testicular feminization syndrome associated with lymphoma is reported. This association has not been previously described. We mentioned an hypothesis about this association.


The special state of the fibril end: site of growth, point of cell surface attachment and possible site for platelet interaction. The end of the collagen fibril is an important and unique morphological site. Growth of fibrils in vitro and in vivo appears to occur by addition of ordered subassemblies of collagen aggregates at the fibril end. In tissues the fibril end appears closely related to the cell surface in both mesenchymal cells and epithelia. In the latter cell type there is an apparent relationship between the basement membrane and fibril end. Platelets do not aggregate upon exposure to basement membranes or collagens derived therefrom. We suggest that the end of the fibril which is embedded in the outer face of the basement membrane may be involved in interaction with the platelet.


The specificity of the reaction of collagen with platelets. 1. Human platelets will react with a number of different collagens from guinea-pig to ostrich. 2. Human skin collagen when treated with pepsin does not react with human platelets. 3. Calf platelets display a different pattern of reactivity than that of human platelets.


Factor XIII, fibrin and collagen.

Factor XIII, fibrin and collagen. It has been demonstrated that activated factor XIII may catalyze the formation of covalent cross-links between fibrin and collagen. This is shown by the disappearance of the gamma-gamma dimer band in PAA-SDS gel electrophoresis when fibrinogen is clotted in presence of collagen, factor XIII and Ca ions, and by the binding of labeled fibrinogen. This reaction may explain the outstanding physiological importance of factor XIII.


[Brain aging. Ultrastructural study of changes in man and laboratory animals]. The authors present a study on ultrastructural changes observed in aged human and animal brain. These changes are analyzed and compared with those described in classic optic microscopy and with those observed in experimental models of the same lesions.


[Brain abscesses. Clinical experience in 27 cases studied by computed tomography]. The prognosis of patients with cerebral abscess depends on an early and accurate diagnosis and its delay carries a high mortality. The CT is at present the diagnostic method of choice for cerebral abscess, because it identifies and localizes precisely the lesion and it allows, based on its morphological characteristic, to select the appropriate time for surgery, and a correct follow up can be easily done. Our experience with 27 cases of cerebral abscess studied by CT is analized and discussed. The mortality rate in our experience was 15%. This is still too high, but we hope and believe it can be reduced with the regular use of CT, appropriate bacteriological studies, and accurate therapeutical management.


[Intracranial pressure. Direct measurement in the cisterna magna]. The intracranial cisternal or supraoccipital punction in the sitting position, was undertaken in 523 cases, of which 329 presented increased intracranial pressure. The procedure must be done by experts, and was well tolerated. There were no cases of death, arterial hemorrhages or medullary lesions. In 451 cases CSF pressure was measured and CSF was extracted for laboratory examination. Pneumoencephalograms were undertaken with this method in 335 patients. The intracranial cisternal pressure in the sitting position is greater than the suboccipital or cervical cisternal pressure by 1.5 to 3cm H2O. Its value is practically equal to the pressure in the posterior fossa, and when the medial ventricular system is patent, is similar to the pressure in the supratentorial fossas. The intracranial cisternal punction has less risks than the suboccipital punction and can be undertaken in patients with rised intracranial pressure In 62 cases the tap was negative; this led to the diagnosis of amygdaline herniation and the procedure was interrupted without mishap. The authors consider that, in the future, the connection of the cisternal catheter with a transducer, will permit the continuous monitoring of the intracranial cisternal pressure.


Dextro-phenylalanine as inhibitor of the noradrenaline release from the isolated rat hypothalamus. In slices of isolated rat hypothalamus, the dextrophenylalanine (d-Phe) 3x10(-3)M produced an inhibition of a 47% in the fractional release of 3H-NA to the preparations of Holtzman rats, and of 39% to the Wistar rats, when the concentration of d-Phe (in this last) was of 1x10(-2)M, pointing that the effectiveness of the d-Phe is variable with the employed rat root. The mentioned effect is calcium dependent, for it was antagonized when the Ca++ was incremented in the medium of 1,68mM (normal) to 2,6mM. Neither atropine, nor phenoxybenzamine interfer the effect to the d-Phe, showing that it will not be due to mechanisms of alpha-adrenergic or muscarinic cholinergic negative feedback. For the moment it cannot be interpretated that the d-Phe effect is performed through specific negative feed-back receivers, for the possible operability of other kind of receptors such as PGs angiotensin, morphinic, etc., has not been discarded yet.


[Immunologic studies in polymyositis syndromes]. Sixteen cases of polymyositis were submitted to study. Out of these sixteen, nine showed no clinical or paraclinical evidences of cancer, and seven presented an associated cancer. In the first group, a normal immune response was observed, whereas in the second group this response was frankly depressed. Emphasis is laid on the significance of the immune response, not only in the diagnosis but as a fundamental factor in prognosis and treatment as well. An early treatment of polymyositis without cancer proved to be satisfactory. On the other hand, when a neoplasm was present, the course of the disease was satisfactorily modified only after the extirpation of the neoplasm.


[Perception and memory of the tactile area in temporal epilepsy]. Tactile perception and memory in epileptics with temporal EEG focus is studied. Out of a total of 24 patients: 12 had right and 12 had left EEG focus. All patients are severe epileptics, not controlled by medical therapy. The Milner and Taylor Model used in a study carried out in commissurotomized patients was followed. We arrived to the following conclusions: There exist disturbances of tactile memory in patients with temporal EEG focus. There is a learning capacity indicating at least a relative conservation of the intellectual capacity. Differences between patients with right and left EEG temporal focus have not been found in this group. Milner and Taylor suggest the normal participation of both hemispheres in this type of task, and that the left hemisphere would add some identifiable and memorable verbal element.


[Clinicopathological analysis of fatal cerebellar hematomas]. An analysis is made of the clinical observations and pathological findings in 16 fatal cerebellar hematomas admitted at the Instituto de Neurología in Montevideo, over a period of 23 years. The absence of definitive lesions of the brainstem in the majority of cases, in spite of the rich symptomatology of brainstem disfunction (horizontal gaze palsies, vegetative syndrome decerebrate movements) is stressed, as well as the rapid evolution of those cases with intraventricular haemorrhage. Missed clinical diagnosis was also frequent in severely ill patients. Discussion is carried out around the importance of early diagnosis in the correct surgical management. Ventriculostomy alone, not followed by immediate craniectomy, may be highly dangerous. Mere evacuation of the cerebellar, and even of the ventricular clots can result insufficient if not followed by ventricular shunting in cases with ventricular inundation.


Photochemistry of rhodopsin and isorhodopsin investigated on a picosecond time scale. Bovine rhodopsin and isorhodopsin were excited with a single 530-nm, 7-ps light pulse emitted by a mode-locked Nd 3+ glass laser at room temperature. Within 3 ps of excitation, absorbance changes due to formation of bathorhodopsin were observed. The difference spectra generated during and 100 ps after pulse excitation are presented. The data show that bathorhodopsin formation is completed within 3 ps for both the primary pigments and suggest that a single common bathorhodopsin is photochemically formed from both primary pigments. Our findings provide additional support for the cis-trans isomerization model of the primary event in vision. Additional absorption transients that were observed near 670 and 460 nm are discussed.


Light scattering vs. microscopy for measuring average cell size and shape. Light-scattering photometry is compared with electron and light microscopy as a source of information about the average size and shape of cells in population. Examined are the effects of limited instrument resolution, necessary experimental procedures, and cell heterogeneity. Information theory is used to survey the relative amounts of information provided by photometric and microscopic measurements. Then in model exploratory experiments, cell size and shape and changes therein are determined both by microscopy and by photometry for spherical and spheroidal cells. Scattering theory is used to calculate photometrically observed light from cell parameters. It is found that if certain other appropriate information about the morphological property of interest is available, then visible light photometry is the preferred method for obtaining quantitative information. It has good absolute sensitivity (0.01-0.10 micrometers resolution), its results are relatively unaffected by sample heterogeneity, it is nondestructive and compatible with many other techniques, it requires no sample preparation, and it provides its information in real time.


Numerical reconstruction of the quantal event at nicotinic synapses. To test our present quantitative knowledge of nicotinic transmission, we reconstruct the postsynaptic conductance change that results after a presynaptic nerve terminal liberates a quantum of acetylcholine (ACh) into the synaptic cleft. The theory assumes that ACh appears suddenly in the cleft and that is subsequent fate is determined by radial diffusion, by enzymatic hydrolysis, and by binding to receptors. Each receptor has one channel and two ACh binding sites; the channel opens when both sites are occupied and the rate-limiting step id the binding and dissociation of the second ACh molecule. The calculations reproduce the experimentally measured growth phase (200 microseconds), peak number of open channels (2,000), and exponential decay phase. The time constant of the decay phase exceeds the channel duration by approximately equal to 20%. The normal event is highly localized: at the peak, two-thirds of the open channels are within an area of 0.15 micrometer 2. This represents 75% of the available channels within this area. The model also simulates voltage and temperature dependence and effects of inactivating esterase and receptors. The calculations show that in the absence of esterase, transmitter is buffered by binding to receptors and the postsynaptic response can be potentiated.


Computer simulation of flow-dependent absorption in microperfused short Henle's loop of rats. With computer simulation we examined the extent to which current theories and experimental data explain function of single microperfused superficial Henle's loops in rats. In the model standard phenomenological equations describe transport; two sets of transport parameters labeled rat and rabbit were taken from published experiments; Michaelis-Menten kinetics in the ascending thick limb were adjusted arbitrarily; tubular radius is either constant or depends on luminal pressure with compliance based on experimental observations; the interstitium is an infinite sink with salt and urea concentrations constant in the cortex and exponentially increasing in the outer medulla; concentrations resemble those found in hydropenic or saline diuretic rats. The following predictions were obtained. The model with rabbit parameters does not recirculate urea and will not operate with high medullary urea concentrations; with rat parameters too much urea recirculates an the results of perfusion with equilibrium solution are not reproduced. Using a compromise between rat and rabbit parameters, the model reproduces water absorption, salt reabsorption, and urea recirculation as observed in vivo in rat loops perfused at 5-40 nl/min. It also simulates perfusion with saline, equilibrium solution, saline plus furosemide, and 300 mM mannitol. When the model includes a short early distal segment, effluent salt concentration reaches a minimum at a 15 nl/min perfusion rate as observed in vivo; however, concentration at the macula densa is a monotonically increasing function of flow. When permeation rate is a function of wall surface area and thickness a better fit to experimental results is produced. However, the effect is small: water absorption alters by 4% or less and effluent salt concentration is reduced by up to 10% at low perfusion rates. Comparison of rigid and compliant loops shows no relationship between transit time per se and reabsorption.


Changes in repair competency after 5-bromodeoxyuridine pulse labeling and near-ultraviolet light. Synchronized V79 Chinese hamster cells, pulse-labeled with 5-bromodeoxyuridine (BrdUrd), show marked changes in the sensitivity to near-ultraviolet light during the cell cycle. Cells are least sensitive during the remainder of the S-phase after the BrdUrd pulse. They become maximally sensitive in the next cell cycle when the BrdUrd-labeled DNA presumably serves as the template for replication. This is followed by a return to relative insensitivity during the remainder of the S-phase, the increase in survival does not occur until DNA synthesis progresses beyond the time when the DNA made during the second pulse serves as a template. Furthermore, cells in the resistant phases of the cell cycle are sensitized by 1-2 mM caffeine. Survival curves are shown for the various cell ages of interest and are discussed in relation to the observed changes in functional repair capacity. The data support the hypothesis that lesions in the BrdUrd-containing DNA are effectively repaired after semiconservative replication. The data indicate that saturation of repair capacity and not target multiplicity is responsible for the appearance of a shoulder on the survival curves.


Null space in the Hodgkin-Huxley Equations. A critical test. Voltage perturbation methods based upon topological concepts are used to elicit responses from the Hodgkin-Huxley (HH) nonlinear differential equations. These responses present a critical check upon the validity of the HH model for electrical activity across squid axon membrane. It is shown that when a constant current is applied such that a stable equilibrium and rhythmic firing are present, the following predictions are inherent in the HH system of equations: (a) Small instantaneous voltage perturbations to the axon given at points along its firing spike result in phase resetting curves (when new phase versus old phase is plotted) with an average slope of 1. (b) A larger voltage perturbation (from certain points along the firing spike) results in the permanent cessation of periodic firing, with membrane voltage rapidly approaching the equilibrium value. (c) A still larger perturbation yields phase resetting curves with an average slope equal to 0. These predictions, coupled with Tasaki's experimental demonstration that squid axons in excellent condition do give repetitive firing under constant current, provide a critical test of the validity of the HH model.


Theoretical shapes of bilipid vesicles under conditions of increasing membrane area. Certain standard properties, including spontaneous curvature, are assumed for the membrane of a bilipid vesicle. Then, if there is a mechanism that causes the membrane area to increase, vesicle cleavage is found to occur.


Flagellar hydrodynamics. A comparison between resistive-force theory and slender-body theory. This paper investigates the accuracy of the resistive-force theory (Gray and Hancock method) which is commonly used for hydrodynamic analysis of swimming flagella. We made a comparison between the forces, bending moments, and shear moments calculated by resistive-force theory and by the more accurate slender-body theory for large-amplitude, planar wave forms computed for a flagellar model. By making an upward empirical adjustment, by about 35%, of the classical drag coefficient values used in the resistive-force theory calculations, we obtained good agreement between the distributions of the forces and moments along the length of the flagellum predicted by the two methods when the flagellum has no cell body attached. After this adjustment, we found the rate of energy expenditure calculated by the two methods for the few typical test cases to be almost identical. The resistive-force theory is thus completely satisfactory for use in analysis of mechanisms for the control of flagellar bending, at the current level of sophistication of this analysis. We also examined the effects of the presence of a cell body attached to one end of the flagellum, which modifies the flow field experienced by the flagellum. This interaction, which is not considered in resistive-force theory, is probably insignificant for small cell bodies, such as the heads of simple spermatozoa, but for larger cell bodies, or cell bodies that have large-amplitude motions transverse to the swimming direction, use of slender-body theory is required for accurate analysis.


DNA damage in synchronized HeLa cells irradiated with ultraviolet. The lethal effect of UV radiation of HeLa cells is least in mitosis and greatest in late G1-early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of UV absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage.


Electrical properties of spherical syncytia. Syncytial tissues consist of many cells whose intracellular spaces are electrically coupled one to another. Such tissues typically include narrow, tortuous extracellular space and often have specialized membranes at their outer surface. We derive differential equations to describe the potentials induced when a sinusoidal or steady current is applied to the intracellular space with a microelectrode. We derive solutions for spherical preparations with isotropic properties or with a particular anisotropy in effective extracellular and intracellular resistivities. Solutions are presented in an approximate form with a simple physical interpretation. The leading term in the intracellular potential describes an "isopotential" cell in which there is no spatial variation of intracellular potential. The leading term in the extracellular potential, and thus the potential across the inner membranes, varies with radial position, even at zero frequency. The next term of the potentials describes the direct effects of the point source of current and, for the parameters given here, acts as a series resistance producing a large local potential drop essentially independent of frequency. A lumped equivalent circuit describes the "low frequency" behavior of the syncytium, and a distributed circuit gives a reasonably accurate general description. Graphs of the spatial variation and frequency dependence of intracellular, extracellular, and transmembrane potential are given, the response to sinusoidal currents is used to calculate numerically the response to a step function of current.


Electrical properties of structural components of the crystalline lens. The electrical properties of the crystalline lens of the frog eye are measured with stochastic currents applied with a microelectrode near the center of the preparation and potential recorded just under the surface. The stochastic signals are decomposed by Fourier analysis into sinusoidal components, and the impedance is determined from the ratio of mean cross power to input power. The data are fit by an electrical model that includes two paths for current flow: one through the cytoplasm, gap junctions, and outer membrane; the other through inner membranes and the extracellular space between lens fibers. The electrical properties of the structures of the lens which appear as circuit components in the model are determined by the fit to the data. The resistivity of the extracellular space within the lens is comparable to the resistivity of Ringer. The outer membrane has a normal resistance of 5 kohm . cm(2) but large capacitance of 10 muF/cm(2), probably because it represents the properties of several layers of fibers. The inner membranes have properties reminiscent of artificial lipid bilayers: they have high membrane resistance, 2.2 megohm . cm(2), and low specific capacitance, 0.8 muF/cm(2). There is so much membrane within the lens, however, that the sum of the current flow across all the inner membranes is comparable to that across the outer surface.


Reference phase analysis of free and bound intracellular solutes. I. Sodium and potassium in amphibian oocytes. A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.


Reference phase analysis of free and bound intracellular solutes. II. Isothermal and isotopic studies of cytoplasmic sodium, potassium, and water. The intracellular reference phase (RP) method and ultra-low temperature micro-dissection were used for isothermal and isotopic phase distribution studies of Na(+), K(+), and water in amphibian oocyte cytoplasm. One-third of the cytoplasmic water is available as solvent for [(3)H]sucrose. This fraction, designated c1, quantitatively coincides with the water volume in which Na(+) and K(+) are freely diffusible. Two-thirds of the cytoplasmic water is inaccessible to sucrose and is designated c2. The Na(+) and K(+) associated with c2 are extremely slowly exchanging (bound) and at different concentrations than in c1. The cations in c1 are in mass-action equilibria with those in c2, each described by an equation of the formC(c) (i) = C(c) (1) (i) + C(c) (2) (i) = q(i).C(RP) (i) + (max)C(c) (2) (i).f(C(RP) (i)in which C(c) (i) is the cytoplasmic Na(+) or K(+) concentration, C(c) (1) (i) is the free, and C(c) (2) (i) the bound cation concentration averaged over the cytoplasmic water. q(i) is the fractional free solute space, C(RP) (i) the RP concentration, (max)C(c) (2) (i) the concentration of binding sites, and the function f is satisfied by the Langmuir isotherm. Numerical values for the variables of the isotherm are determined. Activity coefficients are calculated from RP data and provide a basis for generalizing the oocyte results to other cells. The conclusion is drawn that both c1 and c2 are widely distributed in cells, and that cellular ionic activities involve two distinct systems: the cell-membrane system and an adsorbed water ion-exchange-like buffering system. Alternative explanations for the two-component cytoplasm are considered. A model is proposed in which c1 is a normal intracellular aqueous phase controlled by the plasma membrane, whereas c2 consists of water and ions adsorbed in hydrate crystalline structures. In oocytes these structures are identified with yolk platelets.


Network thermodynamic approach compartmental analysis. Na+ transients in frog skin. We introduce a general network thermodynamic method for compartmental analysis which uses a compartmental model of sodium flows through frog skin as an illustrative example (Huf and Howell, 1974a). We use network thermodynamics (Mikulecky et al., 1977b) to formulate the problem, and a circuit simulation program (ASTEC 2, SPICE2, or PCAP) for computation. In this way, the compartment concentrations and net fluxes between compartments are readily obtained for a set of experimental conditions involving a square-wave pulse of labeled sodium at the outer surface of the skin. Qualitative features of the influx at the outer surface correlate very well with those observed for the short circuit current under another similar set of conditions by Morel and LeBlanc (1975). In related work, the compartmental model is used as a basis for simulation of the short circuit current and sodium flows simultaneously using a two-port network (Mikulecky et al., 1977a, and Mikulecky et al., A network thermodynamic model for short circuit current transients in frog skin. Manuscript in preparation; Gary-Bobo et al., 1978). The network approach lends itself to computation of classic compartmental problems in a simple manner using circuit simulation programs (Chua and Lin, 1975), and it further extends the compartmental models to more complicated situations involving coupled flows and non-linearities such as concentration dependencies, chemical reaction kinetics, etc.


The influence of intercellular clefts on the electrical properties of sheep cardiac Purkinje fibers. A model of a 100 micrometers diameter Purkinje fiber with intercellular clefts was studied under voltage clamp conditions to examine the consequences of radial nonuniformity. Sodium and potassium conductances were distributed so that the surface and cleft membranes had similar channel density. Assuming that the model is appropriate, sodium current (and conductance) measured in the voltage clamp is grossly underestimated because of loss of voltage control of the cleft membrane. Under these conditions a value for g Na of about 15-20 mmho/cm2 of actual membrane is consistent with the experimental measurements of Dudel and Rüdel (1970. Pfluegers Arch. Eur. J. Physiol. 315:136-158.). Intermediate and slow currents (slow inward current and potassium current) appear to be accurately measured under the model conditions, despite some voltage nonuniformity within the cleft. This result depended on the presence of a residual sodium current, and experimental removal of sodium may alter this result. All effects of nonuniformity would be accentuated in fibers of larger diameter.


On the ionic displacement current in lipid bilayer membranes. It is shown that the constant field approximation must be amended to make it apply to time-dependent signals. The necessary additional term corresponds to the ionic displacement current. In the absence of adsorption, this ionic displacement current is found to have a characteristic time of the order of a fraction of a microsecond. We confirm its mathematical form as given by Cole (1965). When the membrane-soluble ions are strongly adsorbed, an additional, purely exponential transient of much larger time constant is calculated, with a time dependence identical to that of the translocation of adsorbed ions. Our results support the pseudostationary approximation used by Andersen and Fuchs (1975) in the description of such exponential transients. Explicit expressions are given for the current after a voltage step as for the admittance, both in the absence and presence of adsorption, for a membrane with a rectangular potential energy profile.


On the adsorption of phloretin onto a black lipid membrane. The effect of uncharged, dipolar phloretin on anion and cation conductance through a black lipid membrane can be used to study its adsorption behavior. The adsorption of phloretin can be described by a Langmuir isotherm with weak dipole-dipole interaction.


A network thermodynamic two-port element to represent the coupled flow of salt and current. Improved alternative for the equivalent circuit. A two-port for coupled salt and current flow is created by using the network thermodynamic approach in the same manner as that for coupled solute and volume flow (Mikulecky et al., 1977b; Mikulecky, 1977). This electrochemical two-port has distinct advantages over the equivalent circuit representation and overcomes difficulties pointed out by Finkelstein and Mauro (1963). The electrochemical two-port is used to produce a schematic diagram of the coupled flows through a tissue. The network is superimposable on the tissue morphology and preserves the physical qualities of the flows and forces in each part of an organized structure (e.g., an epithelium). The topological properties are manipulated independently from the constitutive (flow-force) relations. The constitutive relations are chosen from a number of alternatives depending on the detail and rigor desired. With the topology and constitutive parameters specified, the steady-state behavior is simulated with a network simulation program. By using capacitance to represent the filling and depletion of compartments, as well as the traditional electrical capacitances, time-dependent behavior is also simulated. Nonlinear effects arising from the integration of equations describing local behavior (e.g., the Nernst-Planck equations) are dealt with explicitly. The network thermodynamic approach provides a simple, straightforward method for representing a system diagrammatically and then simulating the system's behavior from the diagram with a minimum of mathematical manipulation.


Enthalpy changes during the photochemical cycle of bacteriorhodopsin. We have used a capacitor microphone calorimeter to measure rapid enthalpy changes that occur when bacteriorhodopsin-containing membrane fragments are excited with short flashes of light. We resolved the enthalpy changes into three phases. At about 100 microsecond after the flash, the bacteriorhodopsins converted into metastable states have an enthalpy about 15-20 kcal mol-1 greater than the enthalpy before excitation. Some of this energy (approximately 10 kcal) is then released to the surroundings as the membrane fragments release protons to the solution. After proton release and before proton rebinding, a large amount of heat is released to the surroundings, equivalent to about 40-45 kcal/mol of bacteriorhodopsin reacting. At this point the energy of the system is about 35 kcal/mol less than it was before the flash; i.e., the system has released all of the energy of the photon (49 kcal/E) plus an additional 35 kcal/mol. Nevertheless, the free energy of the system must still be greater than it was originally, because relaxation to the original state occurs spontaneously. An entropy decrease of at least 125 cal/mol per deg is required to compensate for the heat release. An entropy decrease of this magnitude implies a major increase in molecular order in the purple membrane.


Trajectories of human granulocytes.

Trajectories of human granulocytes. The spatial trajectories of human granulocytes moving on glass in two dimensions under an isotropic medium may be characterized as realizations of a correlated walk. The present model is similar to an earlier one for slime-mold amebae. The correlation from step to step is essentially via the relative angle theta, represented for granulocytes as a random variable with a symmetric bimodal density. As the cells age, they appear to turn less and the degree of correlation increases. Even under chemotaxis the angle theta and the step length r appear to be statistically independent.


A simple tension-displacement model for hemoglobin cooperativity. Based on the Perutz view of hemoglobin cooperativity and the methodology of statistical physics, a molecular model for heme-heme interactions is proposed. The motion of the iron atom with respect to the heme plane is assumed to be the important feature of the oxygenation step, and results in an expression for hemoglobin saturation as an explicit function of the internal tension of the hemoglobin molecule. Closure of the equation is obtained with the assumption of linearity between the internal tension and the displacement of the iron atom above the heme plane. All model parameters are physically realizable and are characteristic of the hemoglobin molecule. Finally, the model is capable of discriminating between positive and negative cooperativity.


Pulse nuclear magnetic resonance measurements of water exchange across the erythrocyte membrane employing a low Mn concentration. A simple, precise, and rapid pulse nuclear magnetic resonance technique for measuring the rate of water exchange across the erythrocyte membrane is presented. The technique is based upon the nonlinear fit of Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation time data of blood doped with 1.7 mM MnCl2 to the general two-compartment exchange condition. Previous approaches using CPMG data required high MnCl2 concentrations (25-53 nM), shown in this work to induce systematic errors ranging from 35 to 45%. At 23 degrees C the average residence time of a water molecule inside the erythrocyte (tau a) is 21.0 +/- 0.6 ms (SE). The Arrhenius plot for water exchange is linear over the range of 3 degrees - 37 degrees C and th Arrhenius activation energy is 4.79 +/- 0.03 kcal (SE). This value does not differ significantly from the energy required for bulk water flow. Results are compared with previous determinations, and sources of systematic error in tau a and the activation energy are evaluated.


The relation of Vmax to INa, GNa, and h infinity in a model of the cardiac Purkinje fiber. The inward sodium current in cardiac muscle is difficult to study by voltage clamp methods, so various indirect experimental measures have been used to obtain insight into its characteristics. These methods depend on the relationship between maximal upstroke velocity of the action potential (Vmax) and the sodium current (INa), usually defined in terms of the Hodgkin-Huxley model. These relationships were explored using an adaptation of this model to cardiac Purkinje fibers. In general Vmax corresponded to INa, and it could be used to determine the relationship of membrane potential to GNa, and h infinity. The results, however, depended on the method of stimulation of the action potential, and an optimal stimulation method was determined. A commonly used experimental technique called "membrane responsiveness" was shown to distort seriously the properties of steady-state gating inactivation that is supposed to measure. Estimation of the changes in maximal sodium conductance, such as those produced by tetrodotoxin (TTX), would be accurately measured. Some experimental results have indicated a voltage-dependent effect of TTX. Characteristics of the measures of TTX effect under those conditions were illustrated. In summary, calculations with a model of the cardiac Purkinje fiber action potential provide insight into the accuracy of certain experimental methods using maximal upstroke velocity as a measure of INa, and cast doubt on other experimental methods, such as membrane responsiveness.


Resonance enhanced Raman spectrum of all-trans anhydrovitamin A. The Raman spectrum of all-trans anhydrovitamin A in hexane at 77 degrees K is presented. The similarity of the Raman spectra of anhydrovitamin A and the protonated Schiff base of retinal is striking. The implications of this for visual pigment studies and bacteriorhodopsin are discussed. Tentative assignments of geometry for four cis-trans isomers of anhydrovitamin A are made on the basis of the observed room-temperature absorption spectra.


Kinetics of carboxymyoglobin and oxymyoglobin studied by picosecond spectroscopy. Picosecond studies of carboxymyoglobin (MbCO) and oxymyoglobin (MbO2) reveal that excitation at 530 nm induces photodissociation at less than 8 ps. The kinetic and structural changes were monitored by following absorbance changes at selected wave-lengths in the Soret (B) band and in the Q band. Within the 10 ps-0.45 ns period of time over which our experiments were conducted, the absorbance changes in the Soret and Q bands for MbCO and MbO2 correspond to the conventional long-term, steady-state deoxymyoglobin difference spectra (Mb-MbCO and Mb-MbO2), as determined by comparison of isosbestic, maximum, and minimum points. In addition, MbCO exhibits a decay to a steady state in the Soret band (monitored at 440 nm). The onset of the decay immediately follows photodissociation and has a rate of (8 +/- 3) X 10(9) s-1 (tau = 125 +/- 50 ps). During the 10 ps-0.45 ns observation window, relaxation is not seen for MbO2 in the Soret band, nor is relaxation observed in the Q band for either MbCO or MbO2. We conclude from these results that the steady state that we observed for MbCO and MbO2 is most likely the stable form of deoxymyoglobin, and the relaxational differences between MbCO and MbO2 observed in the Soret band indicate that the electronic destabilization after ligand detachment is very different for these molecules. We believe that these relaxational differences may be related to differences in tertiary structural changes, or due to the fact that the MbCO (S = 0) molecule passes through an intermediate spin Mb (S = 1) state before relaxing the the Mb (S = 2) state.


Polarized infrared spectroscopy of oriented purple membrane. Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.


Light-scattering effects in the measurement of membrane microviscosity with diphenylhexatriene. Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.


Analysis of picosecond laser induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach. A Pauli master equation is formulated and solved to describe the fluorescence quantum yield, phi, and the fluorescence temporal decay curves. F(t), obtained in picosecond laser excitation experiments of photosynthetic systems. It is assumed that the lowering of phi with increasing pulse intensity is due to bimolecular singlet exciton annihilation processes which compete with the monomolecular exciton decay processes; Poisson statistics are taken into account. Calculated curves of phi as a function of the number of photon hits per domain are compared with experimental data, and it is concluded that these domains contain at least two to four connected photosynthetic units (depending on the temperature), where each photosynthetic unit is assumed to contain approximately 300 pigment molecules. It is shown that under conditions of high excitation intensities, the fluorescence decays approximately according to the (time)1/2 law.


Arsenazo III forms 2:1 complexes with Ca and 1:1 complexes with Mg under physiological conditions. Estimates of the apparent dissociation constants. Experiments to determine the apparent dissociation constants of the Ca and Mg complexes of arsenazo III clearly indicated that the predominant Ca complex contains one Ca ion and two dye molecules, although previous reports have either claimed or assumed 1:1 complexing. The evidence is based on the effects of varying [dye] as well as [Ca] and [Mg], and clear evidence for the formation of 1:1 complexes with Ca was obtained only at submicromolar [dye], whereas Mg formed 1:1 complexes exclusively. The implications of these findings with regard to the use of arsenazo III as an indicator of intracellular free [Ca] are discussed, with particular reference to its selectivity for Ca and the interference effects of other ions.


Pancuronium inactivates alamethicin-induced conductance in artificial membranes. The effect of pancuronium on alamethicin-induced currents was studied in negatively charged lipid bilayer membranes. Pancuronium induces inactivation of the alamethicin-induced current. Inactivation is only observed if this compound is added to the compartment containing alamethicin. Moreover, the process of inactivation is reduced or abolished if pancuronium is added to the alamethicin-free side of the membrane. The time needed to recover from inactivation is greatly reduced if the aqueous solution in the alamethicin-free compartment is stirred. These data suggest that pancuronium permeates through the membrane when the alamethicin-induced conductance is "turned on," binds to the other membrane surface, and changes the surface potential.


The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C. The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.


Electrophoresis and diffusion in the plane of the cell membrane. Electrophoretic and diffusional movements of concanavalin A (Con A) receptors and acetylcholine (ACh) receptors in the plane of the plasma membrane of mononucleate, spherical Xenopus myoblasts were studied by microfluorimetry and iontophoresis. We found that (a) a uniform electric field of 10 V/cm applied along the cell surface produces a partial accumulation of both types of receptors toward the cathodal pole of the cell within 30 min: (b) post-field relaxation of the culture results in the complete recovery of the uniform distribution of the Con A receptors within 10 min; and (c) in contrast to the Con A receptor in general, accumulation of ACh receptors by the electric field results in the formation of stable, localized receptor aggregates. Theoretical analyses were carried out for the distribution of charged membrane receptors at equilibrium between electrophoresis and diffusion, and for the rate of back diffusion after the removal of the field. These analyses indicated that, at 22 degrees C, the average electrophoretic mobility of the electrophoretically mobile population of the Con A receptors is about 1.9 X 10(-3) micron/s per V/cm, while their average diffusion coefficient is 5.1 X 10(-9) cm2/s.


Red cell extensional recovery and the determination of membrane viscosity. A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.


Thermoelasticity of red blood cell membrane. The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol.