The application of thin-layer gel filtration chromatography in the rapid assessment of protein-protein coupling reactions. The use of enzymes as markers when covalently coupled to various antigens or antibodies has wide application in medical science. A variety of bifunctional reagents has been used to produced the conjugates, and there have been some attempts to investigate the conditions of reaction necessary to obtain good coupling, whilst preserving the biological function of the molecules. The present study describes the influence of coupling conditions on the coupling of a alpha-amylase and albumin by a range of commercially available bifunctional coupling reagents, and the application of thin-layer gel filtration chromatography for the rapid qualitative/semi-quantitative assessment of coupling reactions.


Gas-liquid chromatographic method for the measurement of methyl urea in blood and urine. A technique for the measurement of methyl urea in biological fluids is described based upon gas-liquid chromatography of its trifluoroacetyl derivative. The method requires 10 ml of either blood or urine and is capable of measuring methyl urea at concentrations of less than 5 mg/l.


Determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2h-1,4-benzodiazepin-2-one (Ro 7-9957) and its major biotransformation products in blood and urine by electron capture-gas-liquid chromatography. A sensitive and specific electron capture-gas chromatographic assay was developed for the determination of 7-iodo-1,3-dihydro-1-methyl-5(2'-fluorophenyl)-2H-1,4-benzodiazepin-2-one (I) and its major metabolites in blood and urine. The overall recovery of I and its N-desmethyl metabolite (II) from blood is apparently quantitative. The recovery of the major urinary metabolite, the N-desmethyl-3-hydroxy analog (IV), and the minor metabolites, the N-desmethyl analog (II) and the N-methyl-3-hydroxy analog (III) added to urine as authentic reference standards ranged from 80 to 85%. The sensitivity limits of detection are of the order of 2-3 ng of I and 4-5 ng of II per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion pattern in a dog following oral and intravenous administration of a 1-mg/kg dose (total 13 mg), and in man following the intravenous administration of single 5- and 10-mg doses. The N-desmethyl metabolite II was more predominant in dog blood than was the orally or intravenously administered I, but II was barely measurable in human blood.


A specific gas chromatographic method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities. A gas chromatographic (GC) method for the determination of the metabolite resulting from the activities of microsomal styrene monooxygenase (epoxide synthetase) and epoxide hydratase using styrene or styrene epoxide as substrates has been developed. The determination of the activities of both enzymes is based on the GC determination of phenylethylene glycol after its esterification with n-butylboronic acid. Kinetic parameters for both enzymes are given.


Mass fragmentographic quantitation of glutamic acid and gamma-aminobutyric acid in cerebellar nuclei and sympathetic ganglia of rats. A method for the simultaneous quantitation of glutamic acid and gamma-aminobutyric acid (GABA) in tissue by mass fragmentography has been developed. The amino and carboxylic groups of the two amino acids were in a convenient one-step reaction derivatized with pentafluoropropionic anhydride and hexafluoroisopropanol. Deuterium-labeled glutamic acid and GABA and a homologue of GABA have been used as internal standards. The usefulness of the technique has been demonstrated by measurements in parts of rat cerebellum and in the superior cervical ganglion.


High-speed liquid chromatographic determination of phenylpyruvic acid. A high-speed liquid chromatographic method has been developed for the determination of urinary phenylpyruvic acid. This acid is converted by treatment with naphthalene-2,3-diamine into 3-benzyl-2-hydroxybenzoquinoxaline, which is extracted into carbon tetrachloride for separation. 2-Mercaptoethanol is a useful stabilizer, and 2-chlorothioxanthone is a suitable internal standard. The method is specific, and the results are not affected by the presence of such 2-oxo-acids as pyruvic acid, 2-oxobutyric acid, 2-oxoglutaric acid, and 4-hydroxphenylpyruvic acid.


Application of a computer-based chromatograph for automated water pollution analyses. A modified head-space analysis technique, coupling thermal extraction with subsequent adsorption of organics on a poly (para 2,6-diphenyl phenylene) oxide adsorbent (Tenax-GC), has been found to be an efficacious tool when combined with gas chromatography for the study and monitoring of low molecular weight organics present in drinking water supplies (1-4). This procedure has allowed for the analysis of volatile organics in the low parts-per-billion range from 1 liter or less of tap water. Because thermal extraction requires smaller sample sizes than required in carbon-chloroform extraction methods and liquid/liquid extractions, the EPA has recently adopted this type of methodology (5,6). Passage of the Safe Water Drinking Act in December of 1974 has mandated that each state assumes the primary responsibility for carrying out the purposes of the legislation (7). Because the Act says that any supplier of water serving 25 individuals or more must comply with these regulations, large-scale monitoring programs will have to be undertaken. It was thus our desire to try to simplify the sample collection and data acquistion and reduction processes as much as possible, in the effort to make the procedure more adaptable for incorporation into routine monitoring programs. Also, automation reduces the experience and number of personnel required to perform the analyses. In addition to analysis of drinking water samples, the automated procedure described hereinafter can also be applied to monitoring industrial plant effluents, waste water treatment processes, and general quality control monitoring of low molecular weight organic compounds.


Improved column systems for gas-liquid chromatography of iproniazid and iproclozide. As a result of a comparative study performed on thirteen liquid phases an improved set of four analytically packed columns, i.e., Versamid-900, Versamid-930, XE-60, and OV-225 is withheld for successful gas chromatography of iproniazid and iproclozide. Retention data in terms of adjusted retention times and relative retentions, using structurally related compounds as reference, are reported. An evaluation of gas chromatographic performance of proposed columns is described.


Paget's disease of the bone: observations after cessation of long-term synthetic salmon calcitonin treatment. Thirteen patients with Paget's disease of the bone were treated with subcutaneous injections of synthetic salmon calcitonin (SCT) for a mean period of 22 months at doses of 50-100 MCR units daily or 3 times a week. They manifested symptomatic improvement and significant reductions in serum alkaline phosphatase and urinary hydroxyproline excretion during SCT administration. Following discontinuation of SCT, symptomatic improvement was maintained in 10 patients for up to one year, whereas a recurrence of symptoms was seen in only 3 patients. The serum alkaline phosphatase generally showed a return toward pretreatment values 6 months after discontinuation of SCT, whereas urinary hydroxyproline remained depressed for up to a year.


Androsterone sulfate concentrations in plasma in hypo- and hyperthyroidism. Androsterone sulfate concentrations have been measured in the plasma of 16 hypothyroid women and 14 hyperthyroid women by a gas-liquid chromatographic technique and in 23 hypothyroid and 18 hyperthyroid women by radioimmunoassay. In obth studies androsterone sulfate concentrations were significantly higher in the hyperthyroid groups. In one hypothyroid subject, blood was obtained at 20-minute intervals over 24 hours before and after the administration of triiodothyronine; plasma androsterone sulfate, initially 25 mug per dl, increased three-fold as a consequence of treatment.


Dose dependence of growth response to human growth hormone in growth hormone deficiency. A trial of the relative effect on growth of 20 IU/week and 10 IU/week of human growth hormone has been made in 38 patients with "isolated" growth hormone deficiency over 1 year of treatment, 18 patients over 2 years and 10 over 3 years, and in 17 patients with surgically treated craniopharyngiomata over 1 year. The velocity of height growth in the first year of treatment, compared with a full year of pre-treatment control, was 1.3 times as great in both groups of patients on the larger dose as it was in those on the smaller one. Second-degree equations fitted to the treatment catch-up curve gave estimates of 1.7 cm more height gained on the larger dose by the end of the first year, 2.7 cm by the end of the second, and 3.4 cm by the end of the third. Adjusting treatment increment by covariance for bone age at the beginning of treatment, pre-treatment velocity, and body surface area did not alter these mean differences. Bone age velocity during treatment was the same in both treatment groups (mean 1.09 "years"/year in the first year); thus we anticipate a gain in final adult height of the order of 10 cm from employing the larger dose. The decrease in skin folds occurring on treatment, however, was no different with the larger than with the smaller dose. This reinforces previous observations that the short-term metabolic and longer-term auxologic effects of hGH are not necessarily related.


Growth hormone and insulin binding to human liver. Specific binding of 125I-hGH to human liver was found in autopsy specimens from 12 to 15 patients. Specific binding was studied using a new technique employing 20 mu thich "microslices" cut on a cryostat. About 0.5 to 1 mg of tissue per assay tube makes feasible the study of small samples. The range of specific binding was 1.4 to 11.7% of 80,000 cpm 125I-hGH added expressed per mg dry weight of tissue. Specific binding was also demonstratable in homogenates and crude membrane preparations from liver. No correlation was seen between 125I-hGH binding and age, sex, or pathology in the series of patients studied. No specific binding of 125I-hGH was observed in lung, adrenal, spleen, or kidney, although all the tissues bound 125I-insulin. Of several species of growth hormone tested, only primate GH displaced 125I-hGH from its binding sites in human liver. No displacement was seen with ovine or human prolactin or with insulin. Primate placental lactogens had only 0.5-1.0% potency of native hGH in displacing 125I-hGH from human liver. Ungulate placental extracts, however, were equipotent with hGH in this respect.


Triiodothyronine and thyroxine in urine. I. Measurement and application. Urinary triiodothyronine (T3) and thyroxine (T4) were measured by RIA, and T4 was also measured by competitive protein binding (CPB). pH 1-hydrolysable conjugates were 48% of total urinary T3, and enzyme- or pH 1-hydrolysable conjugates were 55% and 61% of total urinary T4. The mean unconjugated T3 excretion was 34.3 ng/h (0.99 mug T3/g creatinine) in normal subjects (no day-night rhythm found), 1.56 mug/g in late pregnancy, 0.82 mug/g in neonates (1-12 days), and was also unchanged in persons with high or low thyroxine-binding globulin (TBG). In thyrotoxicosis, mean T3 excretion was 281 ng/h, no values being in the normal range. In primary hypothyroidism it was 18.3 ng/h, but over half the values were in the normal range. The mean urinary unconjugated T4 was 82.2 ng/h (1.37 mug T4/g creatinine) in normal subjects, 1.6 mug/g in neonates, and unchanged in persons with high or low TBG, except that in pregnancy high values were compatible with increases protein excretion. Apparently increased day-time T4 excretion compared with night-time excretion may also be due to changes in protein excretion rate. The mean T4 in thyrotoxicosis was 337 ng/h (12% of values in the normal range) and 32.8 ng/h in primary hypothyroidism (over half the normal range). All the assays, especially that of T4 by CPB gave readings which were incorrect with protein concentrations above 100 mg/l. Urinary T3 and T4 assays for clinical purposes have few practical advantages over serum assays, despite the relationship of urine T3 and T4 to serum unbound levels.


Triiodothyronine and thyroxine in urine. II. Renal handling, and effect of urinary protein. Mean urinary clearances of T3 were 164 ml/min in normal subjects, 177 in pregnancy, 221 in thyrotoxicosis, 174 in hypothyroidism, and 194 in 3 persons with undetectable T4 but normal T3 levels. T4 clearances were 38 ml/min in normal subjects, 48 in thyrotoxicosis, and 138 in hypothyroidism. Low creatinine clearance was associated with low clearances of T4 and T3. The data suggest urinary excretion of T3 by glomerular filtration of serum unbound T3 with added tubular excretion; and T4 excretion by glomerular filtration of unbound T4 and tubular reabsorption. However, 3-9% of urinary T3 and 5-12% of urinary T4 were bound to urinary proteins, and increased protein excretion caused markedly increased T4 excretion. In addition, 52% of urinary T3 and 68% of urinary T4 were bound to other substances of approximate mol wt 500-2,000, which may influence tubular handling of T3 or T4.


Plasma levels of 5alpha-androstane-3alpha,17beta-diol in boys during adolescent growth. A radioimmunoassay using as antibody against testosterone-3-carboxymethyloxime-BSA is described for the measurement of androstanediol (5alpha-androstane-3alpha,17beta-diol) in human plasma. Sephadex LH 20-column chromatography was used to separate other steroids croos-reaching with the antibody from androstanediol. The sensitivity of the assay was 7 pg and the recovery of labelled androstanediol was 65.1%. The intra-assay coefficient of variation was 9.9%. The existence of 5alpha-androstane-3alpha,17beta-diol in the androstanediol fraction could be demonstrated by gas chromatography and mass spectrometry (GC-MS). Plasma concentration of this substance was measured in 53 normally developing pre-pubertal and pubertal boys and in 13 adult men. The mean concentrations significantly rose from puberty stage 1 to 2, and stage 2 to 3. Although there was no significant difference between the plasma concentrations at stages 3 and 4, an increment from stage 4 and 5 was highly significant. Levels in adult males were significantly higher than those in the stage 5 of normal puberty.


RAdioimmunoassay of human proparathyroid hormone: analysis of hormone content in tissue extracts and in plasma. A radioimmunoassay for human proparathyroid hormone (hProPTH) has been developed and applied to an evaluation of prohormone content in parathyroid tissues and in plasma.


Histological studies of bone from normocalcemic post-menopausal osteoporotic patients with increased circulating parathyroid hormone. Quantitative histological evaluations were made of nondecalcified iliac crest needle biopsies were obtained from 16 untreated, normocalcemic, normophosphatemic postmenopausal osteopenic females. Six of the patients had elevated circulating immunoreactive parathyroid hormone. Morphometric parameters, which were significantly increased in the hyperparathyroid group compared with the euparathyroid patients were the cortical osteoclast count and the percentage of trabecular surface covered by active or inactive osteoid. In addition, in all patients, the cortical osteoclast count, and the per cent of trabecular surface covered by osteoid and inactive osteoid were directly related to levels of immunoreactive parathyroid hormone. These data suggest that progressive osteopenia in some patients with crush fracture, or postmenopausal or senile osteoporosis, may be conditioned by an osteoclastosis, elevations in circulating parathyroid hormone, and a relative increase in poorly mineralized osteoid tissue. As such they emphasize the heterogeneity of a so-called "osteoporotic population" and stress the need for specific histological morphometric evaluation of bone before initiating long-term therapeutic modalities.


Measurements of free thyroxine: comparison of per cent of free thyroxine in diluted and undiluted sera. The effect of dilution on values of the per cent of free T4 in serum (%FT4) measured by equilibrium dialysis, was determined by comparing the values for %FT4 in diluted and undiluted specimens. The specimens studied were sera obtained from 10 normal subjects, 10 patients with hyperthyroidism, 5 with hypothyroidism, and 9 women with normal pregnancy. The method of analysis employed was that described by Sterling and Brenner, except that the 125I-thyroxine used was predialyzed to remove non-thyroxine contaminants and that "corrected" values for %FT4 were calculated by dividing measured values by the numerical extent to which the serum had been diluted.


Pituitary gonadotropin function during human pregnancy: serum FSH and LH levels before and after LHRH administration. Pituitary gonadotropin reserve was evaluated in 8 normal pregnant women (13-35 weeks gestation) by measuring serum concentrations of FSH and LH (betaLH assay) before and after an IV bolus of 100 mug LHRH. Basal levels of FSH and LH were low or undetectable. LHRH administration failed to stimulate FSH release but did result in a small short-lived rise in LH levels. These findings provide further evidence that pituitary gonadotropin synthesis and release are inhibited during pregnancy.


A pressor formation by trypsin from renin-denatured human plasma protein. Trypsin, when incubated with human plasma protein either undenatured or denatured by pretreatment with acid or alkali or by heat, produced an angiotensin II-like pressor substance instead of the depressor bradykinin which has been reported to be formed from various plasma fractions and trypsin. This reaction may be due to the activation of prorenin by trypsin, but this is rather unlikely since it appears that angiotensin II rather than I is formed. It seems to be more likely a direct production of the angiotensin hormone from the substrate, and we propose to call tentatively the active product "tryptensin".


Effect of pyridoxine on human hypophyseal trophic hormone release: a possible stimulation of hypothalamic dopaminergic pathway. A single dose of pyridoxine (300 mg iv) produced significant rises in peak levels of immunoreactive growth hormone GH and significant decrease of plasma prolactin PRL in 8 hospitalized healthy subjects. Serum glucose, luteinizing hormone LH, follicle stimulating hormone FSH and thyrotropin TSH were not altered significantly. In addition, in 5 acromegalic patients who were studied with both L-dopa and pyridoxine, inhibition of GH secretion followed either agent in a similar pattern. These data suggest a hypothalamic dopaminergic effect of pyridoxine.


Preparation and properties of a national reference endotoxin. A large pool of refined endotoxin was prepared from Escherichia coli O113 by extraction with hot acqueous phenol. It was characterized chemically and biologically and will be available for a reference standard designated as reference endotoxin EC.


Ethylenimine-inactivated rabies vaccine of tissue culture origin. The replication of seven rabies virus strains (CVS, HEP, PV, ERA, WIRAB, CPZ and BOLIVAR) in BHK cells and the inactivation dynamics of these strains by beta-propiolactone, acetylethylenimine, and ethylenimine were studied to find the most immunogenic strain and the most economic and stable inactivating agent for the production of an inactivated tissue culture rabies vaccine for animal use. The seven strains reached the peak of virus production 3 to 5 days after inoculation of the cell culture; PV yielded the highest virus titer (10(9) plaque-forming units/ml). The infectivity of virus suspensions containing 10(7) to 10(8) plaque-forming units/0.1 ml was inactivated by beta-propiolactone in 0.5 h, acetylethylenimine in 3.0 h, and ethylenimine in 1.0 h. Most of the vaccine lots prepared with the different strains and inactivating agents passed a modified National Institutes of Health potency test. The vaccines prepared with the PV strain had consistently higher antigenic values (equal or better than four) than the other six strains. This difference was highly significant (F6,12=59.8), whereas there were no statistically significant differences among the antigenic values of the vaccine lots prepared with the three inactivating agents. Batches of lyophilized and liquid vaccine stored at 4 C maintained potency for over 1 year. Ten dogs vaccinated with a vaccine prepared with the PV strain and inactivated with ethylenimine developed a good antibody response and resisted challenge 60 days after vaccination, while seven of eight nonvaccinated controls died of rabies. This information indicates that an inactivated, stable, economic, and easy-to-prepare rabies vaccine can be produced in BHK cells by using the PV strain and ethylenimine as an inactivating agent.


Recovery of pathogenic fungi from clinical specimens submitted for mycobacteriological culture. A total of 332 major pathogenic fungi were isolated from specimens cultured onto both fungal culture media and media used for culturing mycobacteria from January 1968 to June 1975. Only 72 (21.7%) fungi were recovered on media used for culturing mycobacteria. The effect of sodium hydroxide treatment was evaluated and shown to be detrimental to the recovery of fungi. It is recommended that clinical laboratories not rely on mycobacteriological cultures to recover fungal pathogens.


Pseudomonas cepacia strains isolated from water reservoirs of unheated nebulizers. Pseudomonas cepacia strains were isolated from the water reservoirs of unheated nebulizers in a hospital setting. The isolates were characterized by morphology, biochemical tests, and antibiotic susceptibility patterns. An interesting feature of these organisms was their capacity for sustained multiplication in either doubly deionized or doubly distilled water as well as 5% dextrose or 0.9% saline. They could not multiply in doubly deionized and then doubly distilled water or in any of several parenteral nutrition solutions studied. Isolation of Pseudomonas cepacia from the water reservoirs of unheated nebulizers suggests that this equipment may serve as a source of respiratory tract exposure to the organism.


Unusual Chromobacterium violaceum: aerogenic strains. This is the first report of Chromobacterium violaceum strains that are aerogenic. Two pigmented strains of C. violaceum and their nonpigmented variants produced gas in glucose broth. Kliger iron agar, and triple sugar iron agar. They were similar morphologically and biochemically to other anaerogenic strains studied previously.


Isolation and characterization of a catalase-negative strain of Staphylococcus aureus. An unusual, naturally occurring strain of Staphylococcus aureus is characterized. It is typical in colony and cellular morphology, coagulase production, and other biochemical reactions as compared to S. aureus (ATCC-25923), except that it is catalase negative and fails to ferment mannitol anaerobically.


Meningitis due to Aeromonas hydrophila.

Meningitis due to Aeromonas hydrophila. A case of meningitis caused by Aeromonas hydrophila is reported. The infection complicated an otherwise successful frontotemporal craniotomy. Survey of the literature revealed that human infections due to this organism are relatively uncommon, and its causal relationship in meningitis has not been previously reported. A. hydrophila is known to cause bacteremia in patients under treatment with immunosuppresive agents. We report meningitis with bacteremia, caused by this organism, in an immunologically competent host.


Computer-assisted bacterial identification utilizing antimicrobial susceptibility profiles generated by autobac 1. A computer program was developed to identify bacteria solely on the basis of their relative susceptibility to various antimicrobial agents. A sample of 481 clinical isolates from nine of the most commonly isolated gram-negative groups was identified by the quadratic discriminant function technique. Various combinations of antimicrobials were tried, and one set of 18 resulted in a more than 97% correlation with conventional identification procedures. The antimicrobial set could be decreased to 14, while a better than 95% correlation with the conventional procedures was maintained.


Cholesterol requirement of mycoplasmas as determined by microtiter test using polyene antibiotics. A microtiter metabolic inhibition test was used to determine the effect of filipin and lucensomycin on the growth of representative species of Mycoplasma and Acholeplasma. The cholesterol-requiring species tested were found to be very susceptible to the two antibiotics, whereas the cholesterol nonrequiring species were not. The utilization of this method for differentiation between the Mycoplasma and Acholeplasma species is suggested and its advantages are discussed.


Temperature-dependent cultural and biochemical characteristics of rhamnose-positive Yersinia enterocolitica. Clinical isolates of rhamnose-positive Yersinia enterocolitica (Y.e.rh+) were compared with typical rhamnose-negative Y. enterocolitica (Y.e.rh-) and with Yersinia pseudotuberculosis. The Y.e.rh+ differed from the Y.e.rh- and Y. pseudotuberculosis in their ability to ferment raffinose and lactose, utilize citrate and in their inability to grow on Hektoen enteric agar at 22 or 37 C, on Salmonella-Shigella agar at 37 C, and scant on xylose-lysine-deoxycholate agar at 37 C. An extensive temperature-dependent profile of characteristics was established for the Y.e.rh+: motility, acetoin production, citrate utilization, growth on Salmonella-Shigella agar, and ampicillin resistance occurred at 22 C but not 37 C; fermentation of melibiose, raffinose, and cellobiose occurred within 24 h at 22 C, but not before 5 days at 37 C; fermentation of rhamnose and production of beta-galactosidase occurred within 24 h at 22 C, but not before 48 h at 37 C; greater resistance to ampicillin, chloramphenicol, streptomycin, kanamycin, carbenicillin, and gentamicin was observed at 22 than 37 C; and good growth on xylose-lysine-deoxycholate agar occurred at 22 but not 37 C. For optimal recovery of Y.e.rh+ from mixed culture, e.g., stools, two MacConkey plates should be inoculated and incubated, one at 37 C, and one at 22 C. Lactose-negative colonies appearing after 48 h on the 22 C MacConkey agar but not the 37 C MacConkey agar should be considered possible Y.e.rh+. Biochemicals should be tested in duplicate, one set incubated at 22 C, one set at 37 C. Antibiotic susceptibility tests of Y.e.rh+ isolates should be incubated at both 37 C and at a lower temperature to allow the greatest expression of resistance of these organisms to the various antibiotics.


Survival of anaerobic and aerobic bacteria in a nonsupportive gassed transport system. Survival of anaerobic and aerobic bacteria in a commercially available, non-supportive, gassed (oxygen-free) transport container (Anaport) was evaluated quantitatively. Saline-suspended obligate anaerobes survived significantly better in the gassed container in aerobic control tubes (P less than 0.025, t test), and counts were virtually unchanged after 8 h of holding. Similarly, initial counts and relative proportions of a mixture of Bacteroides fragilis and Staphylococcus aureus were maintained for 72 h. The value of the gassed transport system was less apparent when microorganisms were suspended in nutrient broth. The major advantage of the gassed transport system appears to be for holding of specimens collected by saline irrigation.


Anaerobes survive in clinical specimens despite delayed processing. Quantitative cultures were performed on 11 purulent specimens of at least 2 ml from mixed aerobic-anaerobic infections to determine the effect of prolonged exposure to air on the recovery of anaerobes. The specimens were processed immediately and after air exposure for periods of 10 min and 1, 4, and 24 h. There were at total of 37 anaerobic and 36 aerobic strains recovered from these specimens. Of the anaerobes, 26 were isolated with the initial processing and 22 were still present after air exposure for 24 h. The numerical concentrations of anaerobes showed little change with the sequential samplings. Eleven anaerobic strains were not detected in the initial culture but appeared sporadically in subsequent cultures. Using the types of specimens and method of processing employed in this study, most pathogenic anaerobes survived in purulent exudate despite extended periods of air exposure. The major cause of discrepent results with periodic cultures was attributed to vagaries in sampling.


Evaluation of the rapid decarboxylase and dihydrolase test for the differentiation of nonfermentative bacteria. A rapid medium for the detection of lysine and ornithine decarboxylase and arginine dihydrolase activity of 439 strains of gram-negative, nonfermenting bacteria was evaluated and compared with Moeller decarboxylase medium. Results were obtained in 4 to 24 h using the rapid medium, whereas Moeller medium often required extended (3 to 7 days) incubation. There was 100% agreement in the lysine tests with both media and almost 100% agreement in the ornithine tests. There was 91% agreement in the arginine tests, with the significance of discrepant results discussed. The sensitivity, specificity, and quick results obtained by the rapid test make it a suitable substitute for Moeller medium for the identication of gram-negative, nonfermenting bacteria.


Blocking effect of rheumatoid factor and cold agglutinins on complement fixation tests for histoplasmosis. The blocking effect of rheumatoid factor (RF) and cold agglutinins (CA) on the detection of complement-fixing (CF) antibodies for Histoplasma capsulatum using a mycelial (histoplasmin) and a yeast antigen was studied. Sera from 213 patients serologically positive for histoplasmosis were screened for the presence of RF or CA. CF antibodies to H. capsulatum in sera containing RF or CA were studied before and after removal of these factors (RF and CA) by treatment with dithiothreitol. Results suggest that RF or CA may interfere with the CF reaction to the yeast antigen of H. capsulatum but not to the mycelial antigen (histoplasmin).


Evaluation of simplified dichotomous schemata for the identification of anaerobic bacteria from clinical material. Simplified dichotomous schemata are described for the identification of anaerobic bacteria commonly encountered in clinical material. The procedures used are combinations of routine biochemical tests and techniques that are used to uniformly characterize these organisms. Over 200 anaerobic organisms were used in a three-stage evaluation in which data were compared with those obtained by conventional methods. When there was inconsistency between the biochemical tests described in the presumptive identification schemes and gas-liquid chromatography, additional biochemical tests or reference procedures were used to confirm identification. Strains from the American Type Culture Collection and the Center for Disease Control, as well as recent clinical isolates, were included in this evaluation. The results show the simplified procedures to be useful for the identification of anaerobic isolates from clinical material.


Comparative evaluation of the Candida agglutinin test, precipitin test, and germ tube dispersion test in the diagnosis of candidiasis. Normal sera and sera from burned patients were examined for Candida agglutinin titers, precipitin titers, and the ability to disperse germ tubes of Candida albicans in an attempt to determine whether germ tube dispersion is correlated with Candida infection as animal models have indicated. Other investigators have reported that immunoglobulin G antibody to Candida interferes with a serum clumping factor resulting in germ tube dispersion. Germ tube dispersion in sera from burned patients with varying degrees of Candida infection is significantly greater than that found in uninfected controls. In addition, the germ tube dispersion test indicated the presence of Candida infection in several patients who had clinical evidence of infection but no detectable agglutinins or precipitins.


Electron capture gas chromatography study of the acid and alcohol products of Clostridium septicum and Clostridium chauvoei. The metabolic products produced by several strains of Clostridium septicum obtained from patients and animals, along with strains of Clostridium chauvoei, were studied in chopped meat glucose medium by electron capture gas-liquid chromatography (EC-GLC). The strains of C. septicum and C. chauvoei were shown to comprise five different metabolic groups. Both the EC-GLC study and the O and H antigenic study performed previously showed that strains of C. septicum comprise a heterogeneous group. One type of metabolic profile was found only in strains of C. chauvoei. The O antigen types and EC-GLC metabolic types of C. septicum correlated fairly well in isolates from cancer patients but not in stock culture and animal isolates.


Rapid in vitro conversion and identification of Coccidioides immitis. Since many contaminating nonpathogenic fungi resemble Coccidioides immitis culturally, isolates from patients must be inoculated into laboratory animals to confirem identification as C. immitis. This procedure is time-consuming, expensive, and not generally available in clinical laboratories. When cultures of C. immitis were grown in slide cultures on modified Converse liquid medium in purified agar and incubated at 40 C in a candle jar, all 57 isolates demonstrated inhibition of mycelial growth and conversion of arthrospores to endosporulating spherules in 3 to 5 days. Representative isolates of six species of nonpathogenic fungi that resembled C. immitis culturally either did not grow or failed to produce endosporulating spherules under the same conditions. This procedure is recommended for confirming the identication of a culture as C. immitis.


A new medium for the detection of gelatin-hydrolyzing activity of human dental plaque flora. A new medium for the detection of gelatin-hydrolyzing activity of human dental plaque flora by a simplified plate method is described. It is designed to support the growth of obligate anaerobes, facultative anaerobes, and aerobes. This medium can be used for the identification of gelatinase-producing organisms of clinical importance.


Comparison of sodium amylosulfate and sodium polyanetholsulfonate in blood culture media. A comparison between sodium polyanetholsulfonate and sodium amylosulfate in unvented vacuum blood culture bottles containing tryptic soy broth was made with 5,800 sets of blood cultures. No statistically significant differences in isolation rates of bacteria were noted.


Phage typing set for group C1 and C2 Salmonellae. Fifteen common serotypes representative of group C(1) and C(2)Salmonella were characterized using a single set of phages.


Effectiveness of palladium chloride for the isolation of anaerobes. Because of the increased price of palladium chloride, we investigated the effectiveness of the addition of this ingredient to the culture medium used for the recovery of anaerobes. With our system, the addition of palladium chloride did not consistently improve the recovery rate of bacteria from guinea pig intestinal contents and human fecal material.


Prophylactic immunization of humans against rabies by intradermal inoculation of human diploid cell culture vaccine. The antirabies human diploid cell vaccine produced by 1'Institute Merieux, Lyon, France, was administered intradermally to 35 high-risk volunteers using 0.2-ml amounts and various immunization schedules. Three groups never before vaccinated against rabies developed virus-neutralizing antibodies, the titer of which was dose dependent. A single injection stimulated the formation of antibodies. Four inoculations induced the highest antibody levels and the longest persistence of antibody. The administration of a single intradermal booster inoculation was sufficient, even in the case of low-persisting antibody, to elicit a rapid increase of antibodies to high levels. A primary inoculation course of two injections induced a sufficient antibody level which, in case of exposure, could apparently be rapidly elevated by a 0.2-ml intradermal booster inoculation. Adverse side reactions were observed in 7 of 14 individuals after a 1- or 1.5-year intradermal booster inoculation. We therefore suggest that the intramuscular and subcutaneous routes continue to be used for primary vaccinations and that the highly effective intradermal route be restricted to booster inoculations. This is the first long term study of this vaccine and should be a guideline for the pre-exposure treatment of high-risk personnel.


The essentiality of insulin and the role of glucagon in regulating glucose utilization and production during strenuous exercise in dogs. In order to elucidate the role of insulin and glucagon during strenuous exercise (100 m/min, slope 10-12 degrees), we have determined the rates of production (Ra), utilization (Rd), and metabolic clearance (M) of glucose in normal dogs before pancreatectomy and 2 wk after total pancreatectomy (a) when they were being maintained on constant intraportal basal insulin infusion, (245 muU/kg-min) and (b) when insulin supply had been withheld before and during exercise. Such an intense exercise induced in normal dogs a prompt decrease in mean immunoreactive serum insulin (IRI) from 20 +/- 3 to 11 +/- 2 muU/ml. In depancreatized insulin-infused dogs serum IRI during rest and exercise was between 14 +/- 1 and 12 +/- 2 muU/ml. In the third group, after cessation of insulin infusion, IRI decreased by 76% (from 17 +/- 5 to 4 +/- 1) and did not decrease futher during exercise. During exercise, serum immunoreactive glucagon (IRG) increased threefold in normal dogs. In depancreatized dogs serum IRG was the same as in normal resting dogs (indicating a nonpancreatic source of the hormone) but it did not increase during exercise. In normal dogs exercise induced proportional increases in Ra, Rd, and M (threefold) and normoglycemia was maintained. Changes in glucose turnover in depancreatized insulin-infused dogs were similar to those seen in normal dogs suggesting that a decrease in insulin secretion and a rise in IRG are not essential to prevent hypoglycemia in diabetic dogs. With the cessation of insulin infusion in resting depancreatized dogs, Ra increased, M decreased, and hyperglycemia ensued. During exercise, Ra continued to rise, but M did not increase significantly. (a) Regulation of glucose production by liver during exercise is multifactorial. A decrease in IRI and an increase in IRG are not the only factors which can promote delivery of glucose to the peripheral tissues. The insulin glucagon molar ratio was found not to be an essential metabolic functional unit in regulating glucose metabolism during exercise. (b) It is hypothesized that increases in blood flow and capillary surface area can lead to an increase in the amount of insulin delivered to the muscle even when serum levels of IRI are reduced during exercies. It is suggested that small, but adequate amounts of insulin (as found in normal and depancreatized insulin-infused dogs) are essential in regulating glucose uptake in the working muscle. (c) Since totally depancreatized dogs had normal serum levels of IRG (originating presumably from the gastrointestinal tract), the question of essentiality of basal glucagon activity in glucose homeostasis during exercise could not be resolved by these experiments. It appears, however, that regulation of secretion of nonpancreatic glucagon differs from that of pancreatic glucagon.


Is gastrin a major determinant of basal lower esophageal sphincter pressure? A double-blind controlled study using high titer gastrin antiserum. Studies were performed in anesthetized opossums to determine the influence of binding of circulating gastrin with a high titer gastrin antiserum on lower esophageal sphincter pressure. Gastrin antiserum or control antiserum was administered intravenously in successive doses of 0.02, 0.1, and 0.5 ml/kg on separate days. The lower esophageal sphincter pressures were measured for 1 h before and for 1 h after antiserum administration. The control serum caused no binding of opossum circulating gastrin, nor did it modify lower esophageal sphincter pressure. On the other hand, the administration of gastrin antiserum resulted in the binding of 85-90% of circulating gastrin, but it did not reduce sphincter pressure. A continuous infusion of 0.25 mug-kg-1-h-1 of synthetic human gastrin I caused a significant (P less than 0.05) increase in the sphincter pressure, a 30-fold increase in gastric acid output, and a fourfold increase in immunoreactive gastrin in the opossum blood. Prior treatment with 0.1 ml/kg of gastrin antiserum antagonized and 0.2 ml/kg of the antiserum abolished the gastrin-stimulated gastric acid secretion and the stimulating effect of gastrin on lower esophageal sphincter pressure. However, neither dose of antiserum modified basal lower esophageal sphincter pressure. It is concluded that circulating gastrin may be an important determinant of basal sphincter pressure.


Spontaneous in vivo isomerization of bovine serum albumin as a determinant of its normal catabolism. By combination of isoelectric focusing and immunoelectrophoresis of fresh bovine plasma it is shown that 10% of the albumin in plasma has isoionic points equal to the intramolecular SS-interchanged isomers of bovine serum albumin (BSA). It is also shown that (a) albumin with the isoionic point of SS-interchanged BSA is produced in the cow from radioiodinated BSA depleted from SS-interchanged albumin before injection and (b) purified radiolabeled SS-interchanged BSA can be converted in vivo to albumin with the native isoionic point. On this basis, it is proposed that SS-interchanged albumin in vivo is in postsynthetic equilibrium with the "native" albumin conformation. The SS-interchanged isomers purified either from commerical BSA or from BSA submitted to SH-SS interchange was, after radioiodination with 125I, compared metabolically with "native" albumin labeled with 131I in the same calf. Both species of SS-interchanged albumins have fast initial turnover rates but obtain a normal rate of degradation after the reversion to native albumin. If the isomers formed in vivo have the same properties as the ones present in commercial BSA, at least 50% of the physiological degradation of albumin can be accounted for by the 6-7 times faster catabolic rates of these isomers.


Intrarenal mechanisms of salt retention after bile duct ligation in rats. In order to study renal salt-retaining mechanisms during the early stages of ascites formation, rats were subjected to bile duct ligation. After this procedure, plasma volumes were found to be reduced and hematocrits slightly increased. The whole-kidney glomberular filtration rate and plasma flows were reduced to 59 and 57% of control values, but the filtration fraction was unchanged. Absolute sodium excretion, as well as the fraction of the filtered sodium load excreted, was also significantly reduced. When micropuncture techniques were used to examine the function of single superficial nephrons, the glomerular filtration rate in these nephrons was found to be reduced to 70% of controlled values, and fractional reabsorption was found to be increased at all accessible sites along the nephron. Filtration by intermediate and juxtamedullary nephrons, determined by Hanssen's technique, was reduced to 55 and 48% of control values. By the use of radioactive microspheres, it was demonstrated that blood flow to superficial, intermediate, and juxtamedullary nephrons was reduced to 49, 59, and 73% of control values. Filtration by superficial nephrons decreased much more than plasma flow--a finding which suggests that the measured increase in fractional reabsorption was associated with an increase in the superficial nephron filtration fraction. From this study, it appears that two factors play an important part in the sodium retention observed in the initial stages of ascites formation following bile duct ligation in rats: (a) a decrease in the filtered sodium load and (b) increased fractional reabsorption by the superficial nephrons--the nephrons which show the least decrease in filtration.


Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat. The effects of both synthetic and biologically produced angiotensin II (AII) upon the process of glolerular filtration were examined in the plasma-expanded (2.5% body wt) Munich-Wistar rat, by micropuncture evaluation of pressures, nephron plasma flow (rpf) and filtration rate (sngfr). Plasma expansion was chosen as a control condition because (a) response to AII was uniform and predictable, (b) endogenous generation of AII was presumably suppressed, and (c) the high control values for rpf permitted accurate determination of values for the glomerular permeability coefficient (LpA) before and during AII infusion. With subpressor quantities of synthetic Asn-1, Val-5 AII (less than 5 ng/100 g body wt/min), sngfr fell from 47.7 in the control group to 39.8 nl/min/g kidney (P less than 0.005). The rpf fell to 60% of control values (P less than 0.001). Measurement of glomerular capillary (PG) and Bowman's space (Pt) hydrostatic pressures in surface glomeruli with a servo-nulling device permitted evaluation of the hydrostatic pressure gradient (deltaP = PG - Pi). DeltaP increased from 38.1 +/- 1.2 in control to 45.9 +/- 1.3 mm Hg after Asn-1, Val-5 AII and essentially neutralized the effect of decreased rpf in sngfr. The sngfr then fell as a result of a decreased in LpA from 0.063 +/- 0.008 in control to 0.028 +/- 0.004 nl/s/g kidney/mm Hg after Asn-1, Val-5 AII (P less than 0.02). Lower doses of Asp-1, Ile-5 AII (less than 3 ng/100 g body wt/min) had no effect on sngfr, rpf, deltaP, and afferent and efferent vascular resistance, but significantly elevated systemic blood pressure, suggesting peripheral effects on smooth muscle at this low dose. LpA was 0.044 +/- 0.007 nl/s/g kidney/mm Hg after low-dose Asp-1, Ile-5 AII, and 0.063 +/- 0.008 in the control group (0.02 greater than P greater than 0.1). Higher, equally pressor doses of native AII (5 ng/100 g body wt/min) produced effects almost identical to similar quantites of synthetic Asn-1, Val-5 AII upon rpf, deltaP, sngfr, and renal vascular resistance. LpA again fell to 0.026 +/- 0.004 nl/s/g kidney/mn Hg, a value almost identical to that after the synthetic AII. Paired studies with Asp-1, Ile-5 AII also demonstrated a consistent reduction in LpA.


Effects of brief starvation on muscle amino acid metabolism in nonobese man. A reduction in the release of substrate amino acids from skeletal muscle largely explains the decrease in gluconeogenesis characterizing prolonged starvation. Brief starvation is associated with an increase in gluconeogenesis, suggesting increased release of amino acids from muscle. In the present studies, accelerated amino acid release from skeletal muscle induced by brief starvation was sought to account for the accompanying augmentation of gluconeogenesis. To do this amino acid balance across forearm muscles was quantified in 15 postabsorptive (overnight fasted) subjects and in 7 subjects fasted for 60 h. Fasting significantly reduced basal insulin (11.3-7.5 muU/ml) and increased glucagon (116-134 pg/ml). Muscle release of the principal glycogenic amino acids increased. Alanine release increased 59.4%. The increase in release for all amino acids averaged 69.4% and was statistically significant for threonine, serine, glycine, alanine, alpha-aminobutyrate, methionine, tyrosine, and lysine. Thus, with brief starvation, muscle release of glycogenic amino acids increases strikingly. This contrasts with the reduction of amino acid release characterizing prolonged starvation. The adaptation of peripheral tissue metabolism to brief starvation is best explained by the decrease in insulin.


Rapid diagnosis of anaerobic infections by direct gas-liquid chromatography of clinical speciments. Current methods to isolate and identify anaerobic bacteria are laborious and time consuming. It was postulated that the short-chain fatty acids (SCFA) produced by these organisms might serve as microbial markers in clinical material. 98 specimens of pus or serous fluid were analyzed by gas-liquid chromatography, and findings were compared with culture results. Good correlations were found for the recovery of anaerobic Gram-negative bacilli and the presence of isobutyric, butyric, and succinic acids. 19 of 20 specimens with significant amounts of these acids (greater than 0.01 mumol/ml) yielded bacteroides or fusobacteria. Culture of the single "false-positive" specimen failed to grow anaerobic Gram-negative bacilli, although clinical data and Gram-stain suggested their presence. 77 of 78 specimens which has insignificant concentrations of the marker acids failed to yield anaerobic, Gram-negative bacilli in culture. The single "false-negative" specimen yielded Bacteroides pneumosintes, an organism which does not ferment carbohydrates. It is concluded that direct gas-liquid chromatographic analysis of clinical specimens provides a rapid presumptive test for the presence of anaerobic, Gram-negative bacilli.


The effect of inhibition of prostaglandin synthesis on urinary sodium excretion in the conscious dog. Studies were performed to determine the effect of decreased endogenous release of renal prostaglandins on urinary sodium excretion. Two structurally dissimilar inhibitors of prostaglandin synthesis were employed, and studies were performed in conscious dogs allowed to recover from prior surgical instrumentation. Either meclofenamate (2 mg/kg) or the competitive prostaglandin inhibitor RO 20-5720 (1 mg/kg) was given to seven unanesthetized dogs undergoing a water diuresis. The administration of either prostaglandin inhibitor did not alter glomerular filtration rate, renal plasma flow, urinary volume, or potassium excretion. Sodium excretion, however, increased from 32 to 130 mueq/min (P less than 0.02). Essentially, the entire increase in sodium excretion was due to an increase in urinary sodium concentration from 7.7 to 28.3 meq/liter (P less than 0.02). On a different day, the same animals were studied before and after administration of the diluent of the prostaglandin inhibitor. No change was noted in sodium excretion or any other parameter. Thus, these findings suggest that prostaglandin inhibition in the conscious dog is associated with a natriuresis without a change in urinary volume or potassium excretion during water diuresis. This may indicate that the natruiresis was due to diminished sodium reabsorption beyond the distal tubule.


Glucagon response to hypoglycemia in sympathectomized man. Hypoglycemia stimulates immunoreactive glucagon (IRG) secretion and increases the activity of the sympathetic nervous system. To ascertain if the augmented alpha cell activity evoked by glucopenia is mediated by the adrenergic nervous system, the glucagon response to insulin-induced hypoglycemia of five subjects with neurologically complete cervical transections resulting from trauma, thereby disrupting their hypothalamic sympathetic outflow, was compared to six healthy volunteers. In addition to clinical neurological evaluation, completeness of sympathectomy was verified by failure to raise plasma norepinephrine levels during hypoglycemia compared to the two- and threefold increase observed in controls. Total IRG response (IRG area above basal 0-90 min) and peak IRG levels achieved were the same in the quadriplegics and the controls. Although the glucagon rise tended to be slower, and the peak levels attained occurred later in the quadriplegic patients than in the controls, this response was appropriate for their sugar decline, which was slower and reached the nadir later than in the control subjects. These observations that the glucagon release during insulin-induced hypoglycemia is normal in subjects whose hypothalamic sympathetic outflow has been interrupted provide strong evidence that the sympathetic nervous system does not mediate the glucagon response to hypoglycemia.


Nonsuppressible insulin-like activity of human serum. A potent inhibitor of insulin degradation. Nonsuppressible insulin-like activity soluble in acid ethanol (NSILA-s) is a well-characterized peptide derived from human serum which has previously been shown to have insulin-like bioactivity and react with both insulin and NSILA-s receptor sites in liver plasma membranes. In the present study we find that NSILA-s is also a potent competitive inhibitor of the insulin-degrading system of the liver plasma membrane. The most purified NSILA-s preparation tested was 20-fold more potent than insulin itself, and significant inhibition of insulin degradation occurred at concentrations of NSILA-s similar to those found in plasma.


Apoprotein B in fasting and postprandial human jejunal mucosa. We tested whether apoprotein B is present in fasting and postprandial human duodenojejunal mucosa because lipoprotein-like particles are visualized by electron microscopy within the smooth endoplasmic reticulum and the Golgi cisternae of these absorptive cells. Duodenojejunal biopsies from normal volunteers were incubated in citrate buffer and were shaken in 1% EDTA so that absorptive cells could be freed from underlying tissue. Apoprotein B was determined by double-antibody radioimmunoassay in homogenates of absorptive cells. The preparations of absorptive cells were shown to be uncontaminated by plasma lipoproteins; they did not contain any albumin by immunodiffusion able to detect 2 mug/ml. They adsorbed less than 0.1% of 125I-low density lipoprotein which was added to the citrate buffer. Cell preparations from suction biopsies of human rectum contained no detectable apoprotein B. Duodenojejunal absorptive cells from 22 fasting subjects contained 3.2 +/- 0.5 mug of apoprotein B per 100 mg (wet wt) of biopsies or 1.3 mug of apoprotein B per mg of total cell protein. The amount of apoprotein B per milligram of cell protein fell to 0.3 mug in 14 of these individuals whose mucosa was also sampled 45 min after instilling fat intraduodenally. These experiments provide immunochemical evidence that human duodenojejunal absorptive cells contain apoprotein B. This technique should be valuable for studying the physiology of intestinal lipoproteins in absorption and in patients with hyperlipidemia.


Metabolic effects of cloprednol-a new systemic corticosteroid. The short-term metabolic effects of cloprednol, a new short-acting synthetic corticosteroid, were evaluated in four normal subjects. Cloprednol, 12.5 mg/day, in one subject had no appreciable effect on urinary excretion of sodium, potassium, nitrogen, or calcium. Cloprednol, 20 mg/day, in three subjects had no significant effect on mean daily urinary sodium and potassium excretion, although mild, transient sodium retention and kaliuresis were observed. One subject had increased nitrogen excretion and all three had a small increase in calcium excretion. Prednisolone, 40 mg/day, a dose with antiinflammatory potency equivalent to 20 mg/day cloprednol, given subsequently to two subjects under identical conditions resulted in sodium and potassium excretion results similar to those of cloprednol, 20 mg/day, but produced a much greater increase in nitrogen and calcium excretion. These results suggest that, like prednisolone, cloprednol lacks the sodium-retaining properties of hydrocortisone and raise the possibility that cloprednol has less of a deleterious effect on nitrogen and calcium excretion than prednisolone.


Pharmacokinetics of 125I-iothalamate and 131I-o-iodohippurate in man. The pharmacokinetics of 125I-iothalamate (IOT125I) and 131I-o-iodohippurate (OIH131I) have been studied in patients with varying degrees of renal insufficiency. Both compounds have been shown to obey two-compartment model kinetics after intravenous administration. The clearances are closely related to renal function, but poor correlations were obtained between renal function and compartmental distribution characteristics. This study provides further evidence that IOT125I and OIH131I may be used as indicators of renal function in patients after a single intravenous injection.


Fear of trying.

Fear of trying. Unless the nutrition community intervenes in national and international policymaking, the world could well be headed for serious trouble with deaths from starvation on a consequential scale. There has been near-total dissociation between agricultural planning policies and food distribution and nutritional needs of populations. Unfortunately, the nutrition community is out of the mainstream of forces which affect nutritional status and which produce the macro-decisions. Why? Perhaps the professionals have too narrow a view of their role; although malnutrition is a health problem, the solution is not necessarily one of health delivery. Perhaps they don't seriously wish to be involved with policy change and the work it entails. Perhaps, by being unable to achieve consensus within their own ranks, they have generated a lack of credibility. The nutrition community needs to recognize a broader scope for nutrition and to take new approaches in advocacy directed to education policy-makers. No one else can play this role with authority.


Comments on the division of labor in occupations concerned with food. The roles of men and women in relation to food have always been sharply defined: animals belonged to men and plants and food preparation to women. Thus, when the land-grant colleges were established, men were taught animal husbandry and agricultural methods; in home economics, women learned the "home-making" arts, including nutrition and were concerned with people. But, as nutrition became more scientific, men took it over, separating it from home economics. In so doing, less attention was paid to the human elements in food production. World food policies are now made without regard to feeding people, and women have been progressively left out in the decision-making. The increase in nutritional knowledge in the last half century has had little effect on policies. Because Americans set the pattern the rest of the world will emulate, we must come to the realization that we cannot alleviate hunger with money alone-hunger is relieved with food, and food supplies are finite. Real sacrifices in terms of food itself, such as were made in World Wars I and II, will be necessary. Knowledge that millions are truly hungry may convince mothers to feed their children and families in different ways.


Progress toward a national food policy-its implications. Over the last several years, the Senate Select Committee on Nutrition and Human Needs has conducted hearings in an effort to obtain experts' recommendations, prior to proposing legislation to establish a comprehensive national nutrition plan. The framework for such a plan, as developed by the Committee, is presented here. A "nutritional state of the union" report-to include a formal assessment of existing conditions plus specific goals-would be made annually. To insure accountability, a Federal Nutrition Office, headed by a Cabinet-level official, would be responsible for coordination and monitoring all nutrition programs in all governmental agencies and would have the right to review (but not veto) proposals before an agency could issue them officially. In addition, there would be a National Nutrition Center in the Department of Health, Education, and Welfare, charged with day-to-day administration of expanded nutrition program. This Center would operate at the Sub-Cabinet level.


Out-patient dietary management in the Prader-Willi syndrome. Dietary adherence of eight children with the Prader-Willi syndrome was studied in the home environment. Weight changes were recorded at two-week intervals, and measured two-week dietary records were completed twice during the study by the parents of seven of the children. An eighth child was similarly followed for three months, and one dietary record was obtained. Calories, protein, fat, and carbohydrate contents were calculated, and related to recorded weight changes to determine which diets were most practical in controlling weight gain. Caloric requirements of children with the Prader-Willi syndrome appear to be much lower than those of healthy, active children of comparable ages. Age, degree of obesity, familial relationships, and probably, composition of the diet influenced the effectiveness of a given diet. Each family designed a diet which took into consideration the family's eating habits, as well as the needs of the Prader-Willi child. Frequent contact with the dietitian enabled each family to try new food preparation ideas. The effectiveness and acceptability of a low caloric, very low-carbohydrate diet should be tested over long periods in Prader-Willi children whose obesity is being managed in a non-institution setting.


Comprehensive evaluation of fatty acids in foods. VI. Cereal products. Information on total lipid and fatty acid composition or cereal grains and their products used for food has been collated in a comprehensive search of world literature published since 1960. Data considered most suitable for use for representing contents of total lipids and fatty acids have been tabulated and are presented. In developing these data, attention was given to stability and other chemical properties of the lipids, lipid composition of the different parts of the grain, and to such factors as genetics, production, processing, and analytical methods used in extracting and determining the fat and fatty acids in cereal products.


Folklore and food habits.

Folklore and food habits. The folklore surrounding food habits developed from uncertainty and fear in mankind's quest for food. In an attempt to gain control of his destiny, man developed various magical practices which would perhaps assure an abundance of food. The practice and beliefs did not develop haphazardly, but, on examination, reveal a type of "folk logic" explained by Sir James Frazer's concepts of contagious and homeopathic magic. The "logic" was extended to specific practices in preparing foods, eating of foods on special days, the use of food in curing certain diseases, and forbidding foods at certain times. The folk were attempting to coordinate the phenomena of their world according to cause and effect much the same as modern Americans coordinate their world. However, the basic assuptions were different.


Hypochondriasis and the elderly.

Hypochondriasis and the elderly. Hypochondriasis is poorly understood and poorly treated. Attempts to classify it have been unsatisfactory except for Pilowsky's division into primary and secondary groups. Twenty consecutive cases of hypochondriasis in elderly patients were studied; 4 were of the primary type and 16 of the secondary type. For the primary group, neither family therapy nor hospitalization was needed, and the prognosis was good. In contrast, hospitalization was always advised for the secondary group, and such patients improved while in the hospital. Psychotropic drugs, a therapeutic milieu, and often electroshock therapy were needed to treat depression. After discharge, successful treatment necessitated Day Care programs and the long-term use of family therapy. Treatment was unsuccessful in 8 patients characterized by refusal to become involved in post-discharge Day Care programs and family therapy.


Estrogen-androgen levels in aging men and women: therapeutic considerations. The influence of aging on serum levels of gonadotropins (FSH and LH), testosterone and estradiol was studied in the following groups: 4 normal men (ages 30 to 50), 38 men with symptoms of the male climacteric (ages 51 to 84), 25 men with relative impotence (ages 31 to 50), 10 normal women (ages 24 to 31), and 6 menopausal women (ages 58 to 76). FSH and LH levels began to rise in men in their 40's, and the increase became more conspicuous in the later age decades. The degree of elevation was nowhere comparable to that observed in the aging women. In the male, the serum testosterone levels showed a progressive decrease from the fifth age decade onward, whereas in the female there was an increase after the menopause. Estradiol levels showed no significant change in the aged male, but they were somewhat higher than in the aged female. Exceptions to the low-testosterone and low-gonadotropin relationship were observed in individual cases and might be explained by relatively high estradiol values. Proper replacement therapy by means of estrogens for the postmenopausal female and androgens for the aging male is often of great benefit, physically and emotionally.


Treatable aspects of the dementia syndrome. With attentive medical management, the elderly demented patient can live in greater comfort, dignity and independence. Errors in supervision can result in a drug intoxication, misdiagnoses, and personal neglect. Many of the elderly have a chronic dementing illness, and disability often is accelerated by complications. However, some of these complications are treatable. Case histories are presented to illustrate pitfalls in the care of such patients.


Cardiovascular effects of levodopa in aged versus younger patients with Parkinson's disease. The cardiovascular effects of prolonged administration of levodopa were studied in 54 men and women with Parkinson's disease; 23 of them were younger than 70 and 31 were 70 or older. The patients were evaluated clinically before treatment was started and at regular intervals thereafter. The average optimal dosage of levodopa for both age groups was 3.0 and 2.5 gm per day, respectively, during an average treatment period of 20.7 months. Eleven patients showed hypotension (systolic BP of 105 mm Hg or less) that was not related to dosage; in only 6 did the drug have to be permanently discontinued because of syncope; 3 of this group had an associated psychiatric disorder. Four patients had pretreatment hypertension; in 3 the BP fell to normal during therapy; in the remaining patient the hypertension persisted and was successfully treated by an antihypertensive drug. In 5 patients an occasional atrial or ventricular ectopic beat was noted both before and during levodopa therapy but no therapeutic intervention was required. Thirty of the 46 patients with adequate ECG follow-up did not show any significant changes; 5 others showed an increase, and 11 a decrease in myocardial ischemia. Thus the administration of levodopa in elderly patients with or without heart disease is a relatively safe procedure. The only exception would be patients over 70 years of age with a history of previous myocardial infarction. In this group there seems to be a higher incidence of clinically significant hypotension. In such patients, levodopa therapy should be carried out with great caution.


Miles' resection for cancer of the rectum. Miles' resection is the optimal surgical operation for cancer involving the lowermost third of the rectum, although some surgeons question the wisdom of subjecting the patient to permanent colostomy and use fulguration as the alternative. In the author's series of 100 patients, the five-year survival rate was 41.6%. The operative mortality rate was 4%. The median distance of the cancer above the dentate line was 3 cm and the average distance was 4.6 cm. Fifty patients had Dukes' C lesions. Of the 100 patients, 95 had adenocarcinoma, 3 had squamous-cell carcinoma, 1 had a cloacogenic lesion, and 1 had melanocarcinoma. Complications were surprisingly few. Eleven patients had perineal recurrence. The relatively poor survival rate is attributed to anatomic limitations for radical surgical measures and to the spread of cancer cells by the time of operation. Regular check-up examinations are most effective in the prevention of rectal cancer.


An automated microscope for cytologic research a preliminary evaluation. A research-oriented system for automated microscopy is described from an operational point of view. The system consists of a microscope, a TV camera, an automatic cell finder and a servo-driven computer controlled stage. The system is interfaced to a NOVA 840 computer having 112,000 words of 16-bit core memory and extensive peripherals. It is capable of performing a wide variety of image processing tasks and is being used to study various aspects of automated microscopy, with applications in, but not limited to, cytology. Results of preliminary performance evaluations are given.


The use of a slide spinner in the analysis of cell dispersion. A simple and reliable method of determining the degree of dispersion of a cell suspension has been developed using the Perkin-Elmer Uni-Smear Spinner. Optimum conditions regarding rate and duration of spin, etc., were first ascertained using dispersed cell cultures including human cervical cancer cells as well as gynecologic samples. After spinning, single cells in suspension appeared as isolated cells on the slides. Cell aggregates, on the other hand, remained together. Therefore, the distribution of cells in various sized aggregates could be easily quantitated and the slides retained for future review. This method was used to evaluate the dispersing effects of trypsin, ethylenediaminetetraacetate and and syringing human on human gynecology samples obtained by routine cervical scrapes. None of the dispersion methods has, so far, produced an adequate monodispersed cell suspension without unacceptable cell loss.


A real time TV to computer image input system using run coding. Run coding applied to the digitized video signal from a TV scan of cell preparations can effect a substantial reduction in the total amount of data, sufficient to permit a moderate size of store to be loaded within one frame time with a representation of the field adequate for computer analysis. This paper describes the design of a run coding interface between a TV scanner and a computer store which also allows control of scan domain, spatial resolution and density resolution. Results are presented showing its efficiency when dealing with cervical smear preparations.


Coherent optical processing of cervical cytologic samples. Earlier research using a very limited data base gave encouraging results for the automated screening of exfoliated cytologic samples using coherent optical processing techniques to examine individual isolated cells. A more thorough investigation involving a larger data base has confirmed our initial results. This investigation was performed using a specially designed Fourier spectrum analyzer and a solid state optical detector array. An analysis was made to determine the performance of a screening system using such a cell-by-cell discriminating device. This analysis indicated that less than 20,000 cells would have to be examined to obtain a system performance level of 1% false negative and 10% false positive error rates with a 1% probability of occurrence of malignant cells in a malignant sample. This performance figure was inferred from measured statistical performance characteristics of a laboratory cell-by-cell screening device using optically generated Fourier transfrom techniques for cell discrimination. The performance of the system was shown to be much more sensitive to cell-by-cell false error rates than false negative error rates. It was also found that the majority of false positive errors were due to misclassifying parabasal cells as malignant. By eliminating parabasal cells, which comprised more than 25% of our normal cell data base, the number of cells needed to be screened dropped by an order of magnitude. It was also shown that there is an inverse quadratic relationship between the percentage of malignant cells in a malignant sample and the number of cells that must be screened to achieve any desired system performance.


Methods of quantitative autoradiography using incident light microphotometry. Several different approaches of automated grain counting of microautoradiographic grain densities have been reported in the literature. Application of grain counters to cell biology is limited, however, primarily due to shortage of methods allowing the interpretation of grain counts on a molecular basis. Two suitable methods of quantitative autoradiography at the cellular level are reviewed, developed for the isotopes 14C and 125 I. They permit evaluation of absolute radioactivity in autoradiographs and, thus, determination of synthesis processes such as deoxyribonucleic acid synthesis, and of antigen densities on cell surfaces. In this approach towards quantitative autoradiography, grain densities are compared photometrically over labeled cells and over a standard source on the same autoradiograph. Allowance has to be made for the specific geometric factors of the isotope used. This can be advantageously done with an integrating type of measurement using incident light bright-field. With this type of recording, there is an exponential dependence of the photometric values on the radioactive dose. As an example of application, results are presented of the deoxyribonucleic acid synthesis rate of human myelocytes in aplastic anemia and of the immunoglobulin G density on lymphocyte membranes in the normal state and in chronic lymphocytic leukemia.


The hybrid resolution approach to automated autoradiographic analysis. The use of radioactive isotopes in the investigation of cells and their relations to an organism as a physiologic unit is an important area of research to the biologist. The manual analysis of the autoradiographs which result from the use of radioactive tracers is, however, very time consuming and prone to error. This paper describes the utilization of hybrid resolution image processing techniques for the rapid and automatic analysis of tissue region autoradiographs. Nine regional features can be extracted and six properties characterized using the present system implementation. Priliminary measurements on two tissue types show good agreement between manual and automated readings.


A technique for multiple-cell chromosome karyotyping. A new approach to a system for chromosome karyotyping is presented. The system assembles the information about chromosomes from several cells at a time, thereby filtering out noise due to variations in the slide preparations. The system makes i possible to use metaphase spreads which are incomplete due to missing chromosomes, touching and overlapping chromosomes and stain particles. The system gives a precise description of the chromosome complement in terms of distribution function parameters, with the uncertainty of the parameters specificed. The system is adaptive with respect to the initial reference parameter set so that both recognition of normal chromosomes, in spite of the variation displayed among individuals, and identification of aberrant chromosomes are possible. The precise chromosome descriptors can be used to detect differences between the tested individual and various references, in order to find chromosomal abnormalities.


Automatic detection and localization of sister chromatid exchanges. Sister chromatids of human metaphase chromsomes from cells which have replicated twice in medium containing 5-bromodeoxyuridine exhibit unequal fluorescence when stained with the dye 33258 Hoechst. Sister chromatid exchanges occurring in these chromosomes are apparent as interchanges of brightly and dully fluorescing chromatids. A technique for detecting such exchanges by computer analysis of chromsome images has been developed and found to campare favorably with manual methods. The exchanges have been localized in the context of quinacrine banding patterns.


Image processing for automated erythrocyte classification. Digital image processing and pattern recognition techniques were applied to determine the feasibility of a natural n-space subgrouping of normal and abnormal peripheral blood erythrocytes into well separated categories. The data consisted of 325 digitized red cells from 11 different cell classes. The analysis resulted in five features: (a) size, (b) roundness, (c) spicularity, (d) eccentricity and (e) central gray level distribution. These features separated the data into six distinct condensed subgroups of red cells. Each subgroup consisted of morphologically similar cells: (a) macrocytes, (b) normocytes, (c) schistocytes, acanthocytes and burr cells, (d) microcytes and spherocytes, (e) elliptocytes, sickle cells and pencil forms and (f) target cells. The concept of a quantitative "red cell differential" was introduced, utilizing these subgroup definitions to establish subpopulations of red cells, with quantifiable indices for the diagnosis of anemia, at the specimen level.


Performance of the LARC classifier in clinical laboratories. As part of the installation procedure of the LARC leukocyte differential classifier in a clinical laboratory, a 100-slide protocol is carried out to establish the performance of the classifier in the laboratory. The detailed make-up of this protocol and its relationship to key performance parameters for the leukocyte differential are described in detail. Data from the first ten of these protocols are presented which establish the (a) normal ranges, (b) reproducibility, (c) accuracy, (d) false-positive/false-negative rates for the detection of left shifts and (e) false-positive/false-negative rates for the detection of bloods with abnormal cells.


Basic principles of electrical sizing of cells and particles and their realization in the new instrument "Metricell". To understand the basic events during the passage of particles through the Coulter orifice, three experiments were performed. (1) The denpendence of the volume results on the particle path has been shown by ink-colored particle beams. (2) The deformation and alignment of cells during their passage through the orifice have been photographed by a nano-second photographing technique. (3) The absolute volume evaluation of particles has been studied with model particles in enlarged model orifices of different lengths. A new compact sizing instrument, "Metricell," equipped with a particle-independent electrical calibrating system, is described.


The computer analysis of volume distribution curves. Demonstration of two erythrocyte populations of different size in the young guinea pig and analysis of the mechanism of immune lysis of cells by antibody and complement. Guinea pig, rat and sheep erythrocytes were sized electrically using the hydrodynamic focusing technique. The experimental curves were approximated with a computer by linear and logarithmic normal distributions. Rat and guinea pig erythrocytes from adult animals were best approximated by one linear normal distribution. Two populations (I, II) of erythrocytes with different mean volume could be demonstrated in young guinea pigs by this analysis. Population I erythrocytes are small, have a lower electrophoretic mobility and are mainly present at birth. They are gradually replaced by the larger population II erythrocytes. Both types of erythrocytes are probably the result of separate differentiation pathways. The analysis of erythrocyte volume distribution curves during immune lysis by antibody and complement shows that intact and ghost erythrocytes are measured by electrical sizing. No volume changes were observed up to the EAC1-8 intermediate. After the addition of C9, a C9 dose-dependent part of the erythrocytes swell permanently to spheroids. The spheroid transformation is a temperature-dependent, all or nothing reaction which is independent of protein osmotic forces from the interior of the cell.


Comparison of techniques improving the resolution of standard Coulter cell sizing systems. Latex spheres with nominal diameters of 1.857, 2.02, 3.5, 5.2 and 9.69 mum were used to compare improvement in Coulter resolution gained (a) by a simple hydrodynamic focusing modification and (b) by Coulter edit and high resolution circuits. Since pulse size profiles of focused 9.69-mum spheres appeared quasimonodisperse [geometric SD (GSD) congruent to 0.016 congruent to (1 + Arithmetic SD/arithmetic mean)], unfocused profiles of these particles approximated the error function resulting from inhomogeneities in the electric and hydrodynamic sensing-zone fields. As expected, this field error function differed systematically with differences in orifice diameters (30, 50, 70 and 100 mum) and length to diameter ratios (0.75 or 1.2), the error being reduced slightly by the edit circuit and considerably by the high resolution circuit. Data on the 1.857- and 2.02-mum spheres (focused GSDs congruent to 1.06) and 3.5- and 5.2-mum spheres (focused GSDs congruent to 1.6) indicated that the field error affected closely similar particles similarly but its influence on the unfocused population means and variances differed in the GSD regions of 1.06 and 1.60.


Estimation of cell size from pulse shape in flow cytofluorometry. The fluorescence pulse widths (pulse duration) generated by fluorochromed cells in a flow-through cytofluorometer provide useful information regarding cell (or nuclear) size and possibly other morphologic features. Simple fixed thresholds just above background noise can be used to identify these pulses, but measurements are then strongly affected by random noise and will vary as a result of both pulse amplitude and pulse shape. In this paper, we propose two alternative, amplitude-independent estimates of pulse width. The first is based on a threshold at some fraction of pulse height, or on a pair of thresholds scaled to some fixed central fraction of the total integrated intensity. The second is based on the ratio of pulse area to peak height. The quantitative properties of these width estimators is studied with simulated fluorescence pulses and with experimental specimens of fluorchromed polystyrene spheres, pollen and spores of known different diameters. The results indicated that absolute particle diameters can be measured within a precision of approximately 1 mu using instruments for flow cytofluorometry.


Cell discrimination by multiangle light scattering. Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.


Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells. A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.


Pulse-height light-scatter distributions using flow-systems instrumentation. Several laboratories have recently been making light-scatter measurements on cells and other particles using flow-systems instrumentation. We at the Los Alamos Scientific Laboratory, as well as others, have obtained multimodal pulse-height distributions in certain angular regimes from particles of supposedly uniform characteristics. Because it was assumed that multimodal distributions implied characteristics of multivalue, the accuracy of such data has been doubted. In the present work, pulse-height distributions anticipated on the basis of exact electromagnetic theory were calculated for particles of known characteristics. These calculated pulse-height distributions agree quite well with those obtained experimentally. Physical optics form the basis for the explanation of the complex pulse-height distributions obtained experimentally. However, the results of this study show that certain cautions are necessary in the interpretation of light-scatter data presented in this manner.


Slit-scan cytofluorometry. Basis for automated prescreening of urinary tract cytology. A study was undertaken to assess the applicability of the slit-scan technique to automated prescreening of urinary tract cytology. Cells from voided and catheterized urines were stained with acridine orange and measured on a static cell slit-scan cytofluorometer. Analysis of data from the specimens indicates that nuclear fluorescence alone appears adequate for recognition of abnormal specimens. Remaining problems in the automation of urinary tract cytology prescreening are discussed.


Flow microfluorometric system for screening gynecologic cytology specimens using propidium iodide-fluorescein isothiocyanate. Seventy cervical cytology specimens have been screened by a xero resolution flow analyzer-sorter using propidium iodide and fluorescein isothiocyanate as fluorochromes for nucleus and cytoplasm, respectively. This system shows a 1% sensitivity for detection of abnormal cells using only crude visual data analysis. Screening of clinical specimens was performed on the instrument with a 5.8% false negative rate and a 11.8% false positive rate by comparison with routine visual cytologic evaluation of the same samples.


Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata. The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.


Purification of the chromosomes of the Indian muntjac by flow sorting. Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.


Assessment of monocyte esterase activity by flow cytophotometry. An azo dye supravital method has been devised for selectively staining human monocytes in suspension for nonspecific esterase activity. Stained cells can  be identified and rapidly enumerated by presenting the suspension of stained cells to the Cytograf, a flow-through cell discriminating cytophotometer. The intensity of stain is proportional to the intracellular esterase activity. By analysis of the oscilloscope display, it has been possible to obtain relative data concerning the degree of activity of monocyte nonspecific esterase activity. These observations suggest a unique approach to the measurement of intracellular enzyme activity in selected cells in a mixed population.


Automated analysis of antigen-stimulated lymphocytes. Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and tuberculin-purified protein derivative to determine if flow microfluorometric techniques had the sensitivity to detect high and low levels of blastogenic response. The deoxyribonucleic acid content of the lymphocytes was analyzed after culture. Blastogenic response was found to be measurable both sensitively and reproducibly. Tritiated thymidine incorporation measurements were also made. Preliminary experiments using inactivated Herpesvirion (type I) as a stimulant showed blastogenic response detectable by flow microfluorometric deoxyribonucleic acid measurements. Refinements of this technique may prove to be useful in the study of lymphocyte response to viral antigens in patient populations.


Quantitation of lymphocyte response to antigen by flow cytofluorometry. The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.


A generalized machine for automated flow cytology system design. A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.


Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins. Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.


Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of deoxyribonucleic acid. Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.


Quantitation of fluorescence fading phenomena for identifying intracellular biopolymers. The fading behavior of the 670 nm fluorescence emission band produced by unfixed rat mast cells stained with acridine orange (AO) has been found to be in excellent agreement with the behavior predicted by second order chemical kinetics. The reciprocal of fluorescence intensity plotted against time yields a straight line. When due account is taken of dye/cell ratio and the intensity of fluorescence-exciting radiation, Io (measured with the standard phosphor particle), the slope of this straight line is a constant, k'', which is independent of dye/cell ratio and Io. k'' differs from the second order photochemical rate constant by a constant factor. The fading of a given AO-biopolymer complex is described by a particular value of k''. Two values of k'' have been found for rat mast cell granules, indicating the presence of two different AO-biopolymer complexes. Fading of fluorescence may serve to identify particular intracellular biopolymers in individual cells even when present in a heterogeneous population.


Quantitation of the rate-zonal sedimentation spectrum. Rate-zonal sedimentation of cells from the female genital tract has been employed as a means of separating many of the different cellular components from each other. Ultra-slow streak photography of forward scattered light from the sedimentation spectrum has been used as a monitor of the success of the individual sedimentation maneuver. The presence of convention currents vitiates the separation potential of the technique, and this is readily detected by the photographic monitor. In addition, these photographs clearly indicate the positions of bands of cellular entities of different sedimentation velocities. Further study of the quantitative aspects of the light scattered from each part of the spectrum reveals that it possesses a quantitative representation of the concentration of cells at each level of the spectrum. The photographs and densitometric scans support these expectations. To avoid the limitations of film as a data-logging medium and to obtain real-time system utilization, a direct photoelectric scanner has been assembled. Preliminary tests indicate ready feasibility.