High performance liquid chromatographic determination of nine phenolic antioxidants in oils, lards, and shortenings.

A simple, rapid technique is described for the determination of 2- and 3-tert-butyl-4-hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), 2,4,5-trihydroxybutyrophenone (THBP), propyl gallate (PG), octyl gallate (OG), dodecyl gallate (DG), and nordihydroguaiaretic acid (NDGA) in vegetable oils, lards, and shortenings. The antioxidants are partitioned from hexane-oil into acetonitrile, concentrated under vacuum, and determined by reverse phase, gradient elution, high performance liquid chromatography with detection at 280 nm. The mobile phase is water-acetonitrile with 5% acetic acid. With this system, only Ionox-100 and OG are not resolved. Recoveries from soya-sunflower seed oil spiked at 16 and 100 ppm and from lard at 32 ppm for 8 of the 9 antioxidants ranged from 96 to 103%, 100 to 102%, and 98 to 102%, respectively. The recoveries of BHT, due to incomplete extraction, were 84, 85, and 87%, respectively.


Microbiological determination of penicillin G, ampicillin, and cloxacillin residues in milk.

A fast cylinder plate microbiological method was developed for the quantitative determination of penicillin G, ampicillin, and cloxacillin in milk. Agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis were used and incubated at 64 degrees C for 4 1/2 hr. Standard curves were obtained for the following ranges of concentration of antibiotics: 0.004-0.064 IU penicillin G/mL, 0.0025-0.04 microgram ampicillin/mL, and 0.03-0.48 microgram cloxacillin/mL. The method is suitable for detecting penicillin residues in milk and for quantitative milk-out studies of the above antibiotics used in treatment of bovine mastitis.


Mycotoxins in animal feedstuffs: sensitive thin layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone, and T-2 toxin.

Improvements have been made to a previously described multi-mycotoxin method that involved a membrane cleanup step. Using 2-dimensional thin layer chromatography and appropriate solvent systems, aflatoxin B1 can be detected in mixed feedstuffs and various ingredients at levels ranging from 0.1 to 0.3 microgram/kg. Corresponding detection limits for ochratoxin A and sterigmatocystin are 5 to 20 microgram/kg and for T-2 toxin and zearalenone 20 to 200 microgram/kg.


Gas-liquid chromatographic/mass spectrometric screening method for T-2 toxin in milk.

A method for the analysis of T-2 toxin in milk is presented. Ethyl acetate extracts of milk samples which had been spiked with T-2 toxin were purified by thin layer chromatography and derivatized with N,O-bis(trimethylsilyl)acetamide to produce the T-2 toxin trimethylsilyl ether (T-2 toxin-TMS). N,O-bis(trimethylsilyl-d9)acetamide was used to make T-2 toxin d9-trimethylsilyl ether (T-2 toxin-d9 TMS) which was added to the derivatized milk extract as an internal standard. Samples were analyzed by combined gas-liquid chromatography/mass spectrometry using either electron impact ionization or chemical ionization mass spectrometry. In electron impact ionization analyses, simultaneous monitoring of the T-2 toxin-TMS fragment ion at m/z 436 and the T-2 toxin-d9TMS fragment ion at m/z 445 gave a T-2 toxin-TMS detectability estimated at 6 microgram/kg. In chemical ionization analyses, the T-2 toxin-TMS fragment ion at m/z 377 and the T-2 toxin-d9TMS fragment ion at m/z 386 were simultaneously monitored to give a T-2 toxin-TMS detectability estimated at 3 microgram/kg. Average recovery was 85% at 200 microgram/kg and 65% at 20 microgram/kg.


Comparative study of two methods for extraction of aflatoxin from peanut meal and peanut butter.

The difference between the CB and Best Foods methods in extracting aflatoxins from peanut products has been studied. The CB method yields 60, 121, 35, and 22% higher results for aflatoxins B1, B2, G1, and G2, respectively for 4 samples of peanut meal and 6 samples of peanut butter studied. Both reverse phase liquid chromatography and thin layer chromatography were used to quantitate the extracted aflatoxins.


High pressure liquid chromatographic determination of carbohydrates in food products: evaluation of method.

A high pressure liquid chromatographic (HPLC) method for determining 5 common carbohydrates in food products was evaluated. Reproducibility data were generated showing a relative standard deviation of 2.8%. Recovery studies on a variety of foods gave an average recovery of 98.8%. The HPLC data for 3 varieties of ready-to-eat cereals were compared with data from 4 independent laboratories using current AOAC chemical methods. The HPLC mean values differed from the chemical mean values by 3.2%.


High pressure liquid chromatographic determination of the rodenticide brodifacoum in rat tissue.

An HPLC method was developed to determine residues of individual isomers of brodifacoum (3-[3-4'-bromo[1,1'-biphenyl]-4-yl)-1, 2, 3, 4-tetrahydro-1-naphthalenyl]-4-hydroxy-2H-1-benzopyran-2-one) in rat tissue. The compound was extracted twice with 10% methanol in chloroform, filtered, and cleaned up by using automated gel permeation chromatography. A final cleanup on a silica gel SEP-PAK was added to protect the analytical column from irreversible adsorption and to reduce analysis time. The analysis was done on a microPorasil column; a UV detector was used for quantitation. Recoveries of brodifacoum added to rat tissue in concentrations of 0.12-5.0 ppm were greater than 90%.


Gas-liquid chromatographic determination of permethrin in bovine tissues.

A method is presented for the determination of permethrin (m-phenoxybenzyl cis,trans-(+/-)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) in bovine tissues. Fat and muscle samples were cleaned up first by liquid-liquid partition on a bonded phase chromatographic column. Final cleanup of fat and muscle was performed on a short Florisil column. Liver, kidney, spleen, and heart tissues only required cleanup on a Florisil column before quantitation of permethrin by electron capture gas-liquid chromatography.


Quantitative determination of tris-(2,3-dibromopropyl) phosphate by densitometry of thin layer chromatograms.

The flame retardant tris-(2,3-dibromopropyl) phosphate (Tris) was chromatographed on silica gel thin layer plates, using ethyl acetate-hexane (30+70) as the developing solvent. Tris was visualized by spraying the chromatogram with 1% aqueous silver nitrate followed by exposure to ultraviolet light for 40 min. Tris appeared as dark gray spots on a white background; the spots were quantitated by densitometry at 660 nm. The lower level of sensitivity was 50 ng; calibration plots were linear from 50 to 800 ng. The procedure was developed to determine the levels of unmetabolized Tris remaining after exposure to sewage sludge suspensions. Recovery of Tris from 3 sewage sludge samples fortified at the 1.0 ppm level averaged 97%.


Analytical characteristics of late-eluting halogenated flame retardants.

The analytical characteristics of halogenated flame retardants were studied. Suitable electron capture gas-liquid chromatographic conditions were developed, and retention times and sensitivity responses were obtained. The behavior of these industrial chemicals through the AOAC multiresidue method for nonfatty foods was studied using fish as the test substrate. Of the 24 flame retardants investigated, 23 were recovered (85-110%) using the AOAC method.


Separation of normal paraffins and evaluation of petroleum contamination in foods.

A method for separating n-paraffins from petroleum hydrocarbons in foods was developed. The method consists of 5 initial steps: digestion of sample with alkali, silica gel column chromatography, molecular sieve adsorption, destruction of the sieve with HCl, and oxidation with KMnO4. Recoveries of n-paraffins added to 55 g oyster at a level of 0.36 ppm ranged from 80% for normal pentadecane to 100% for n-paraffins over 18 carbon atoms. This method also facilitated the analysis of iso-paraffins such as pristane (2,6,10,14-tetramethylpentadecane) and phytane (2,6,10,14-tetramethylhexadecane), and other hydrocarbons, which are thought to be good marker compounds for the estimation of petroleum pollution.


Colorimetric determination of uric acid in poultry excreta and in mixed feeds.

Uric acid was determined in samples of poultry excreta and animal feeds to which poultry excreta were added. The rapid colorimetric method used by Van Handel was adapted for this purpose. The method is simple, accurate, and gives reproducible results.


Screening method for vitamins A and D in fortified skim milk, chocolate milk, and vitamin D liquid concentrates.

Vitamin A was determined in fortified chocolate milk and skim milk; vitamin D was determined in fortified chocolate milk, skim milk, and vitamin D concentrates, using reverse phase high pressure liquid chromatography (HPLC). The sample is saponified, extracted with hexane, and chromatographed in an HPLC system on a 10 micron Vydac TP reverse phase C18 column, using acetonitrile-methanol (9+1) as the mobile phase. For 6 replicates, the recoveries of vitamins A and D, using this procedure, were 99 and 98%, respectively.


Distribution of aflatoxins B1 and M1 in contaminated calf and pig livers.

The determination of aflatoxins B1 and M1 in multiple sections of livers from 4 calves and 1 pig exposed to high levels of aflatoxins showed a uniform distribution of the aflatoxins in each liver, within the precision of the analytical method used. The thin layer chromatographic method has an expected within-laboratory coefficient of variation of 15%.


Teaching autistic children to use sign language: some research issues.

Three questions are raised with respect to the use of sign language as an alternative system of communication for nonverbal autistic children. First, does teaching a child to sign facilitate speech development? The data suggest that following simultaneous communication training, mute children are not likely to learn to talk; however, a combination of simultaneous communication training and separate vocal training may have a synergetic effect on speech development. In contrast, children who initially have good verbal imitation skills apparently show gains in speech following simultaneous communication training alone. Second, what is the upper limit of sign acquisition? Data suggest that abstract concepts, syntax, and generative skills can be taught. Procedures used in the operant conditioning of speech may prove useful in training complex signing skills. Third, does sign acquisition result in a general improvement in adaptive functioning? It appears that following sign training, some children do show increases in spontaneous communication, decreases in self-stimulatory behavior, and improvement in social skills. However, these outcomes are often difficult to interpret. Some data are described that help clarify the relationship between sign training and general behavioral improvement.


The simplest treatment alternative: the law of parsimony applied to choosing appropriate instructional control and errorless-learning procedures for the difficult-to-teach child.

A method for choosing effective teaching procedures for difficult-to-teach children is proposed. Assessment of child responses during teaching that involves gradually increasing environmental support in the learning setting is the basis for choice. The levels of environmental support in which child responses are assessed are (1) trial-and-error procedures; (2) increased environmental support involving analyses of reinforcement systems, incompatible responses, and prerequisite skills, as well as the most effective use of instructional control; and (3) errorless-learning procedures. Effects of instructions upon learning are discussed in terms of instructional detail and pacing, as well as with respect to the role of instructions in feedback and progressively delayed cue procedures. Stimulus shaping and stimulus fading are discussed in terms of the effectiveness of each for teaching children who have difficulty learning with more traditional procedures. The importance of the incorporation of criterion-related cues when utilizing stimulus shaping or fading is emphasized. It is proposed that an assessment of child responses should be made with respect to the three general levels of environmental support, as well as from sublevels within these, in order to choose the simplest but still effective alternative procedure for teaching difficult-to-teach children.


The effects of schedule of reinforcement on stimulus overselectivity in autistic children.

Recent research demonstrated that when autistic children are presented a discrimination task with multiple cues, they typically respond to an abnormally limited number, usually one, of the available cues. This phenomenon, termed "stimulus overselectivity," has been implicated as a possible basis for many of the behavioral deficits characteristic of autism. The present investigation was conducted to systemically analyze the effects of changing the schedules of reinforcement during discrimination training on subsequent stimulus overselectivity. Twelve autistic children were taught a discrimination involving multiple visual cues, with a CRF schedule of reinforcement. The children were then overtrained on either the same (CRF) schedule or on a partial (VR:3) reinforcement schedule. Subsequent overselectivity on single-cue test trials was then assessed. Results suggested that significantly less overselectivity occurred when the children were presented with the VR:3 reinforcement schedule during overtraining. These results are discussed in terms of variables influencing overselectivity and in terms of implications for designing treatment procedures for autistic children.


A food satiation and oral hygiene punishment program to suppress chronic rumination by retarded persons.

Food satiation and oral hygiene punishment were used to treat the non-life-threatening rumination of two institutionalized profoundly retarded persons. Satiation consisted of allowing the clients to eat until a satiation criterion of food refusal was achieved or until two full meal portions were consumed. The oral hygiene procedure consisted of cleansing the clients' teeth and gums with Listerine for 2 minutes following each instance of rumination. In the formal study, three conditions--baseline, satiation, and satiation plus oral hygiene--were used following the lunch meal in a multiple-baseline across-subjects design. One client's rumination decreased from an average of 89.5% during baseline to 48.8% during the satiation condition and to 3% during satiation plus oral hygiene. The second client's rumination decreased from a baseline average of 49.9% to 7.9% during satiation and to 1.4% during satiation plus oral hygiene. Generalization probes taken following the breakfast and dinner meals showed a systematic decline in rumination as the various conditions were implemented following the lung meal. In the 16-week follow-up, rumination was treated following all meals with oral hygiene, and satiation was used at one of the daily meals for 1 week on a rotating basis. Rumination remained at a near-zero level following all meals throughout the follow-up. Thereafter, a maintenance program was conducted by the ward staff. The satiation plus oral hygiene punishment treatment program appears to be an immediate, effective, enduring, and humane method of treating the non-life-threatening rumination of retarded individuals.


Purification and characterization of 3-hydroxyacyl-CoA dehydrogenase of Mycobacterium smegmatis.

3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel filtration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereospecificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i.e., 6.0 for the oxidation of NADH and 9--10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i.e., esters of CoA, pantetheine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4 mM, respectively. Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The antibodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.


Purification and properties of alkaline phosphatase from the mucosa of rat small intestine.

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume,  B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.


Fate of newly synthesized histones shortly after interruption of DNA replication.

The synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on polyacrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C]marker histones by a 15 min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hydroxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hydroxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.


An improved method for the preparation of chondroitin by solvolytic desulfation of chondroitin sulfates.

By heating the pyridinium salts of chondroitin 4- and 6-sulfates in dimethyl sulfoxide containing 10% of water or methanol at 80 degrees C for 1--5 h, several chondroitin preparations with sulfur contents of 0.02--1.05% were prepared in 83--96% yields, respectively. Chemical properties of the preparations, such as degrees of desulfation and of depolymerization, were compared with those of the products obtained by the previously described methods.


Asymmetric manipulation of the membrane lipid bilayer of intact human erythrocytes with phospholipase A, C, or D induces a change in cell shape.

Changes in the membrane morphology and phospholipid content of human erythrocytes were determined after incubation of intact cells with each of various exogeneous phospholipases (PLases). PLase A2 from Naja naja or bee venom induced crenation of the cells in parallel with hydrolysis of the membrane phosphatidylcholine (PC). This crenated cell shape was reversed to a biconcave disc or cup-like form by a further treatment with lysophospholipase. In contrast, bacterial PLase C from Clostridium perfringens and Pseudomonas aureofaciens or fungal PLase D from Streptomyces chromofuscus induced invagination of the cells in parallel with hydrolysis of the PC. The action of the latter group of PLases on the membrane morphology was counteracted by PLase A2, and vice versa. Thus, participation of the membrane lipid bilayer in the induction of membrane conformational change and hence cell shape change was demonstrated.


Effect of butyrate on glycolipid metabolism of two cell types of rat ascites hepatomas with different ganglioside biosynthesis.

The effect of butyrate on glycolipid metabolism and morphological differentiation in the cell culture system of rat ascites hepatomas, AH 7974 of island-forming type and AH 7974F of free type, was studied. Both cell lines adhered to the substratum in the presence of 1 mM butyrate. In the case of AH 7974, the addition of butyrate induced a distinct morphological change but the other cell line showed no such conspicuous change. Butyrate-treated AH 7974 cells showed a 2 to 3-fold elevation of CMP-N-acetylneuraminic acid: lactosylceramide sialyltransferase activity to form N-acetylneuraminylgalactosylglucosylceramide (GM3). On the other hand, no enzyme activity could be detected in AH 7974F cells. Four glycosyltransferase activities involved in glycolipid synthesis, including sialyltransferase in AH 7974F cells, were reduced by butyrate. From these observations we concluded that sialyltransferase to form GM3, or TM3 itself, is prerequisite for the morphological alteration induced by butyrate.


Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture.

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.


Addition of short guanylyl blocks to oligonucleotide primers with a thermophilic polynucleotide phosphorylase. Its application to the synthesis of oligonucleotides containing guanylyl residues.

Polynucleotide phosphorylase from Thermus thermophilus catalyzed the addition of short guanylyl blocks from GDP to the 3'-hydroxyl termini of oligonucleotide primers at low temperature in a simple reaction mixture. Polyguanylic acid formation was inhibited at 37 degrees C, but the addition of one or two guanylyl residues to oligonucleotide primers proceeded in high yields. The reaction was applied to the synthesis of oligonucleotides containing guanylyl residues at the 3'-end. Using (Ap)2A and (Up)2U as primers, (Ap)3G, (Ap)3GpG, and (Up)3G were synthesized in yields of 25--52%. (Ap)2GpG was synthesized from ApA and GDP in a yield of 13%.


Purification and characterization of a lectin from rice bran.

A rice bran lectin was purified to homogeneity by precipitation with ammonium sulfate and chromatography on ovomucoid-Sepharose and CM-cellulose. The molecular weight of the dimer lectin was estimated to be around 37,000 by ultracentrifugation studies. The sedimentation coefficient was 3.8S. On Sepharose 6B gel filtration in the presence of 6 M guanidine-HCl, the lectin showed a molecular weight of 19,000. On reduction and carboxymethylation, the lectin further dissociated into two nonidentical subunits, with molecular weights of about 11,000 and 8,000. These subunits did not show hemagglutinating activity. Equilibrium dialysis experiments using N-acetyl-[1-14C]glucosamine indicated that about 1.8 mol of the sugar was bound to 19,000 g of the lectin. The lectin was mitogenic against mouse splenic lymphocytes and human peripheral lymphocytes. The lectin enhanced the rate of glucose oxidation and inhibited epinephrine-stimulated lipolysis in mouse adipocytes. Some characteristics of the lectin are compared with those of wheat germ agglutinin.


Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals.

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.


Isolation and characterization of L-fucose dehydrogenase from rabbit liver.

L-Fucose dehydrogenase [EC 1.1.1.122] was isolated from a rabbit liver extract and purified about 390-fold with a yield of approximately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 celluose colum chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sephadex 4B, was useful for the removal of other dehydrogenases. The eznyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.7 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.


Difference UV-absorption spectrum of scallop adductor myosin induced by ATP.

A difference UV-absorption spectrum induced by ATP was observed with scallop myosins purified from both striated and smooth adductor muscles. The difference spectra showed a positive peak at 289 nm with a shoulder around 295 nm, and two small negative troughs around 280 nm. Tryptophanyl movement similar to that in rabbit skeletal myosin is indicated. Some tyrosyl movements in scallop myosins, however, may be in the opposite direction.


Thymidine kinase enzyme variants in Physarum polycephalum. Kinetics and properties of the enzyme variants.

Thymidine kinase variants of Physarum polycephalum separated by repeated DEAE-cellulose chromatography have been characterized. The enzyme variants show similar catalytic properties (e.g., substrate specificity, pH optimum) and molecular weights, as judges by their sedimentation in sucrose gradients. However, they differ significantly with respect to pI, inhibition by dTTP and thermostability, and they have slightly different Km values for deoxythymidine as a substrate.


A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column.

Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.


Ganglioside compositions of erythrocytes from various strains of inbred mice. Ocurrence of sialosylgalactosylceramide in red blood cells of inbred mice.

The gangliosides from the erythrocytes of various strains of inbred mice were analyzed. The ganglioside compositions differed in different strains and the strains could be divided into 4 types on the basis of this difference. The main ganglioside is GM4 or sialosyl galactosyl ceramide in Type 1, and unidentified ganglioside in Type 2, GM4 and GM2 in almost equal amounts in Type 3, and GM2 in Type 4.


Lipogenesis in Ehrlich ascites tumor cells under anaerobic culture conditions.

Anaerobic culture conditions (95% argon/5% CO2) caused a slightly greater increase in total lipids of Ehrlich ascites tumor cells than a gas phase of 20% O2, 75% N2, 5% CO2. Whereas the rate of [U-14C]acetate incorporation into total lipids and lipid-subclasses rose markedly in the absence of oxygen, a drastic decrease of [U-14C]pyruvate and [1-14C]octanoate incorporation as well as a 30% reduction of 3H incorporation into lipids from tritiated water were observed under these conditions. Since profound changes in the metabolic state of cells cause alterations in the specific activity of the acetyl-CoA pool but do not alter the specific activity of intracellular water, this precursor is considered to be an adequate monitor for lipogenesis under aerobic and anaerobic culture conditions. Therefore, it is concluded that Ehrlich ascites tumor cells are not able to reoxidize NADH/NADPH in the absence of oxygen by a stimulation of biosynthesis of fatty acids as is discussed to be the case in normal cells. The slight increase in total lipids of anaerobically cultured cells seems to be the result of an imbalance between normal uptake and impaired utilization of lipids from serum-supplemented culture medium.


[Comparative investigations on the organotropic carcinogenic effect of different N-nitroso compounds with rat after single and chronic treatment (author's transl)].

After the gavage of 200 mg N-nitrosodiethylamine per kg body weight only kidney tumors developed while long-term administration of 10 ppm N-nitrosodiethylamine induced esophageal tumors and hepatocellular carcinomas in female rats (SIV 50). This change of the organ-specific carcinogenic effect is not observed in experiments with N-methyl-N-nitrosobenzylamines substituted with a methyl group at the phenyl moiety. Both chronic treatment and single doses induced tumors of the esophagus and the pharynx.


[The influence of cytostatic and immunosuppressive methyl hydrazones on myelo- and lymphopoiesis in vitro (author's transl)].

The cytostatic and immunosuppressive N'-methyl-N'-beta-chloroethylbenzaldehyde hydrazones B1 and B2 inhibit the colony growth of mouse bone marrow cells and PHA-stimulated human lymphocytes in vitro in a dose-dependent manner. In the presence of B1, however, in contrast to B2, the inhibition of 3H-thymidine (3H-Tdr) uptake by the bone marrow cells and lymphocytes is insignificant. Two further benzaldehydrazones CyB4 and EB4 show little or no influence both on clony growth and nucleoside uptake. On the other hand, CyB4 inhibits the 3H-Tdr uptake by ConA- or LPS-stimulated mouse spleen cells to a gretaer degree than does B1 or B2, although CyB4 unlike B1 or B2 does not display any immunosuppressive effects in the mouse. These findings demonstrate that the 3H-Tdr method is less sensitive than the colony assays and is hence only of limited value as a measure of the vitro proliferation of mammalian cells treated with cytostatics.


Spontaneous endocrine tumors in Sprague-Dawley rats.

Spontaneous endocrine tumors were found in 81 of 100 Sprague-Dawley rats (42 males and 39 females) which survived for more than 2 years. Most of these tumors were medullary carcinomas of the thyroid, followed by tumors of the anterior pituitary gland, pheochromocytomas and cortical adenomas of the adrenal gland, and islet cell tumors of the pancreas. Multiple occurrence of these tumors was frequently observed. This study describes the morphology of these spontaneous endocrine tumors.


Kinetochore microtubule numbers of different sized chromosomes.

For three species of grasshoppers the volumes of the largest and the smallest metaphase chromosome differ by a factor of 10, but the microtubules (MTs) attached to the individual kinetochores show no corresponding range in numbers. Locusta mitotic metaphase chromosomes range from 2 to 21 mum, and the average number of MTs per kinetochore is 21 with an SD of 4.6. Locusta meiotic bivalents at late metaphase I range from 4 to 40 mum(3), and the kinetochore regions (= two sister kinetochores facing the same spindle pole) have an average of 25 kinetochore microtubules (kMTs) with an SD of 4.9. Anaphase velocities are the same at mitosis and meiosis I. The smaller mitotic metaphase chromosomes of neopodismopsis are similar in size, 6 to 45 mum(3), to Locusta, but they have an average more kMTs, 33, SD = 9.2. The four large Robertsonian fusion chromosomes of neopodismopsis have an average of 67 MTs per kinetochore, the large number possibly the result of a permanent dicentric condition. Chloealtis has three pairs of Robertsonian fusion chromosomes which, at late meiotic metaphase I, form bivalents of 116, 134, and 152 mum (3) with an average of 67 MTs per kinetochore similar to Locusta bivalents, but with a much higher average of 42 MTs per kinetochore region. It is speculated that, in addition to mechanical demands of force, load, and viscosity, the kMT numbers are governed by cell type and evolutionary history of the karyotype in these grasshoppers.


Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.


NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii.

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro-synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.


Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.


Ultrastructure of double minutes from a human tumor cell line.

Double minutes (dm) have been isolated from human tumor cells by zonal centrifugation and by differential pelleting of chromosome suspsension. These methods allowed collection of dm in sufficient quantity and purity for visualization with electron microscopy. Ultrastructurally, the chromatin fibers in dm resemble thrance fragments was found. When the two isolation protocols were compared, differential pelleting was shown to increase purity twofold to 85% dm by mass. The differential pelleting procedure enables easy collection of dm in sufficient quantity and purity for chemical analysis.


Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and P1.

Three clinical patterns of inhibition of MgATP2--activated flagellar motility have been found by measuring the motility of Triton-demembranated sea-urchin spermatozoa beating with their heads attached to a glass surface. Inhibition of beat frequency by the reaction products, ADP and Pi, is competitive with the normal substrate, MgATP2-, and the inhibitory effects are similar to a reduction in MgATP2- concentration. Inhibition of beat frequency by ATP4- is competitive with MgATP2, but is accompanied by an inhibition of bending, as measured by the angle between the straight regions on either side of a bend, which is not seen when MgATP2- concentration is reduced. Inhibition of beat frequency by Mg2+ is not competitive with MgATP2-, and is accompanied by an increase in bend angle, so that there is no change in the rate of sliding between flagellar tubules. These differences suggest unexpected complexity of dynein ATPase action in flagella. The beat frequencies of both swimming and attached spermatozoa show a linear double reciprocal dependence on MgATP2- concentration, with identical slopes. The calculated sliding velocities between tubules also give linear relationships, but the slopes are different, suggesting that beat frequency may be the more fundamental dependent variable in this system.


Synthesis of intracellular membrane proteins in vitro. Relation between rough endoplasmic reticulum and mitochondrial outer membrane.

Hepatic rough microsomes were incubated in a messenger-dependent protein-synthesizing system from rabbit reticulocytes. Up to 30% of the total product labelled with [35S]methionine, and subsequently recovered with the microsomes, was located in an intrinsic protein fraction associated with these membranes, i.e. was retained by the membrane following extensive sonication in the presence of 1.5 M KCl, 0.1% deoxycholate, and 5 mM ethylenediaminetetra-acetate (EDTA). When products synthesized with the use of membrane-free mRNA from rough microsomes and free polysome were post-incubated with rough microsomes, ribosome-stripped rough microsomes, or outer mitochondrial membrane, low amounts of intrinsic-type polypeptide product were recovered with these membranes. Higher recovery was achieved, however, when ribosome-stripped rough microsomes were added at the beginning of polypeptide synthesis in a reticulocyte lysate supplemented with additional ribosomal-wash factors. Analysis of these products by polyacrylamide gel electrophoresis showed that a number co-migrated with intrinsic proteins located in both rough microsomes and mitochondrial outer membrane. In addition, a prominent in vitro product co-migrated with a major protein which is located in outer mitochondrial membrane fractions, but is barely detectable in rough microsomal fractions. The present experiments were unable to detect a unique set of intrinsic polypeptides which were synthesized and assembled in vitro under the direction of mRNA from free polysomes, and not from rough microsomes. The results suggest that synthesis of at least some intrinsic membrane proteins which are destined for the outer mitochondrial membrane occurs on rough ER in rat liver.


Stem cell growth and differentiation in Hydra attenuata. I. Regulation of the self-renewal probability in multiclone aggregates.

Interstitial stem cells in Hydra are rapidly proliferating multipotent stem cells which continuously give rise to precursors for nerve and nematocyte differentiation. Growth of the stem cell population is controlled by the cell cycle time of the stem cells and the self-renewal probability, Ps (the fraction of stem cells in each generation which divide to yield more stem cells). In normal Hydra the stem cell generation time is 24 h and Ps = 0.6; under these conditions the stem cell population doubles in 3.5 days. In the present experiments we have systematically investigated the dependence of Ps on stem cell density. We culture stem cells in a feeder layer system consisting of aggregates of nitrogen-mustard (NM)-inactivated Hydra cells. In this system stem cell density can be varied over a wide range by changing the number of clone-forming units (CFU) added to the aggregates. We have measured the growth rate of the stem cell population and the cell cycle of stem cells in NM aggregates after 4--7 days of culture. From these data we calculate the value of Ps. The results indicate that the growth rate decreases 4-fold as the number of CFU seeded per aggregate increases from 10 to 400. Under these same conditions the cell cycle remains constant. The values of Ps calculated from these results indicate the Ps decreases from 0.75 in aggregates seeded with 10--30 CFU to 0.55 in aggregates seeded with 200--400 CFU. These results support a model in which Ps is controlled by negative feedback from neighbouring stem cells. In addition, our experiments indicate that Ps decreases during the growth of stem cell clones. When only a few stem cells are seeded in aggregates, they give rise to isolated clones distributed throughout the aggregate. Ps decreases markedly within such clones as they grow in size presumably due to increasing stem cell content of the clones. Since Ps in such isolated clones declines with growth, we infer that the local stem cell concentration is what controls Ps and that the spatial range of the negative feedback signal is short compared to the dimensions of NM aggregates.


Stem cell growth and differentiation in Hydra attenuata. II. Regulation of nerve and nematocyte differentiation in multiclone aggregates.

The differentiation of nerve cells and nematocytes from interstitial stem cells in Hydra has been investigated under conditions of changing stem cell density. Interstitial stem cells were cultured in a feeder layer system consisting of aggregates of nitrogen mustard-inactivated tissue. The aggregates were seeded with varying numbers of stem cells from 10 to 400 per aggregate; between 4 and 7 days later the rates of nerve and nematocyte differentiation were measured. Nerve differentiation was scored by labelling the stem cell population with [3H]-thymidine and counting nests of 4 proliferating nematoblasts. In both cases the numbers of differentiating cells were normalized to the size of the stem cell population. The results indicate that the rate of nematocyte differentiation increases as the concentration of stem cells increases in aggregates; under the same conditions the rate of nerve differentiation remains essentially constant. To calculate the numbers of stem cells entering each pathway per generation, a computer was programmed to simulate the growth and differentiation of interstitial stem cells. Standard curves were prepared from the simulations relating the rates of nerve and nematocyte differentiation to the fraction of stem cells committed to each pathway per generation. The rates of nerve and nematocyte commitment were then estimated from the experimentally observed rates of differentiation using the standard curves. The results indicate that nerve commitment remains constant at about 0.13 stem cells per generation over a wide range of stem cell concentration. Nematocyte commitment, by comparison, increases from 0.15 to 0.21 stem cells per generation as stem cell concentration increases in aggregates. The fact that the ratio of nerve to nematocyte commitment changes under our conditions suggests that stem cell commitment is not a stochastic process but subject to control by environmental stimuli.


Ammonia plus another factor are necessary for differentiation in submerged clumps of Dictyostelium.

Differentiation of Dictyostelium amoebae can occur in submerged clumps of cells; under an oxygen atmosphere mature stalk cells and spores form, as has been shown in previous work. This report shows that at least 2 factors are released by the cells under these conditions, and that both, together, are required for differentiation of stalk cells and spores. One of the factors is ammonia (NH3 + NH4+). The other factor(s) is heat stable and dialysable but has not yet been further characterized. The factors can be collected in conditioned medium and, when added to cells, stimulate differentiation. Conditioned medium loses its biological activity upon the removal of the NH3 + NH4+. When NH3 + NH4+ is added back, activity is restored. Because NH3 + NH4+, alone, has no activity, a second factor(s) in the conditioned medium must be required for differentiation. It is also shown that calcium inhibits differentiation in submerged clumps and that in calcium-free medium the timing of differentiation is essentially the same as under aerial conditions.


Immaturity substances in Paramecium primaurelia and their specificity.

Cells of Paramecium primaurelia in the immaturity period of the life cycle contain substances which repress the expression of mating ability when introduced into mature cells. When the immature cytoplasm of P. primaurelia was injected into mating-reactive cells of P. tetraurelia, P. caudatum and P. multimicronucleatum, immaturity was also induced in them. But the immature cytoplasm of P. primaurelia was ineffective when injected into mature cells of P. bursaria. When the immature cytoplasm of P. bursaria was injected into mating-reactive cells of P. primaurelia and P. tetraurelia, no difference from the control was observed. When the immature cytoplasm of P. caudatum was injected into mating-reactive cells of P. primaurelia and P. tetraurelia, immaturity was induced in cells of the latter 2 species.


An abnormality of cell behaviour in human dystrophic muscle cultures: a time-lapse study.

The initiation of monolayer mass cell cultures from adult human biopsies has revealed a striking abnormality in the growth and development pattern of muscle cultures from Duchenne-type dystrophy. This abnormality in cell behaviour was seen as early as 4 days in culture, well before myotube formation or confluence, and consisted of areas where cells clustered together in a multilayered mass rather than showing the typical monolayer distribution normally observed. To gain some insight into the mechanism of cell cluster development, we have examined such a culture by time-lapse cinematography and also the cell behaviour of other control cultures. The results of this study show that the clusters enlarged primarily by cell division and, to a lesser extent, by the acquisition of neighbouring cells. Furthermore, none of the single cells surrounding the clusters exhibited contact inhibition of movement. This behaviour was not observed in the other cultures examined. These findings could be indicative of an abnormality in the cell surface or cell-locomotory machinery of dystrophic cells.


The sorting out of embryonic cells in monolayer, the differential adhesion hypothesis and the non-specificity of cell adhesion.

It has been reported previously that sorting out of chick embryonic liver parenchyma and limb bud mesenchymal cells would take place in monolayer culture. The distribution of cell types obtained (liver formed the internal, discontinuous phase) was interpreted in terms of the differential adhesion hypothesis. It was suggested that, in monolayer, liver cells were more cohesive than limb bud cells. In this paper we set out to extend the previous observations with 2 particular questions in mind: (i) Is sorting out in monolayer a general phenomenon occurring between a wider range of cell types? (ii) Can evidence be provided for or against the interpretation of results in terms of the differential adhesion hypothesis? Sorting-out experiments were conducted on circular hydrophilic islands, on an otherwise hydrophobic substratum. Under these conditions, sorting-out in monolayer was obtained with binary combinations of 4 chick embryonic tissue types: liver parenchyma, limb bud mesenchyme, pigmented epithelium of the eye and corneal epithelium. With every combination but one, the cells of one type surrounded the cells of the other type, generating what we have called a 'circle-within-a-circle' configuration. With the remaining combination, liver parenchyma and corneal epithelium, only localized sorting was obtained. The 'circle-within-a-circle' configuration is consistent with an interpretation in terms of the differential adhesion hypothesis, according to which the distribution of cells is determined by the relative strengths of cohesions between their lateral surfaces. In direct support of this is the finding from plating the different cell types at sub-confluent density on hydrophilic substrata that limb bud is the cell tye having the weakest lateral cohesion in monolayer. Limb bud surrounded the other 3 tissues on hydrophilic island. A hierachy of lateral cohesiveness between the 4 cell types has been constructed. It is unlikely that the results can be explained in terms of specific cohesion. When plated together at subconfluent density, the 3 epithelial cell types aggregate together to form mixed monolayered islands, suggesting that they share common adhesive mechanisms.


Different mechanisms in the attachment of cells to native and denatured collagen.

The attachment of cells to collagen has been reported previously to require the presence of serum and the particular serum protein involved in this process, variously known as CIG, CAP or fibronectin, has been isolated. This conclusion that cell attachment to collagen requires serum (or more precisely, fibronectin) is based on experiments measuring the kinetics of cell attachment to films of collagen. We have measured the kinetics of attachment of HeLa and attachment to films of collagen-containing substrata under a variety of experimental conditions and present evidence that the serum-dependent mechanism of cell attachment described by others is actually only the case for films of denatured collagen, while cell attachment to native collagen fibres occurs by a different, serum-independent, mechanism. The possible relevance of these findings to cell-matrix interactions in vivo is discussed.


Cell surface lipids and adhesion. IV. The effects of trypsin on lipid turnover by the plasmalemma.

Trypsin treatment of intact cells or isolated plasmalemmae from embryonic chick neural retinae leads to an accumulation of lysophospholipids in the plasmalemmae. Trypsin was used at activities commonly used in cell disaggregation techniques. This accumulation appears to result from the decrease in acyltransferase activity in the plasmalemma produced by enzyme treatment. Plasmalemmal CoA ligase activity is not affected by trypsin treatment. Trypsinization has little effect on plasmalemmal phospholipase A2 activity. These results are discussed in relation to (a) the effects of trypsinization on cell adhesion, and (b) the theory that cells cannot adhere to lecithins because of their fluidity or surface-free-energy values. We propose that the effects of trypsinization on adhesion may in large part be due to the effects on other plasmalemmal proteins. Similarly the inability of cells to adhere to lecithin substrates is simply explained as being due to the lysolecithin that contacting cells release from these substrates.


A lectin which binds specifically to beta-D-galactoside groups is present at the earliest stages of chick embryo development.

Extracts obtained from chick embryos at the pre-gastrula and gastrula stages are able to agglutinate trypsinized rabbit erythrocytes fixed with glutaraldehyde. Agglutination is inhibited by saccharides sharing a beta-D-galactopyranoside configuration. Agglutinin activity is also inhibited by desialysed fetuin which bears terminal galactose residues but not by native fetuin, desialysed agalacto-fetuin, bovine submaxillary mucin and desialysed bovine submaxillary mucin. Lectin activity is present in extracts obtained from the embryonic area of the blastoderms as well as in extracts from extra-embryonic endoderm and ectoderm. In extracts subjected to gel filtration on ECD Sepharose, lectin activity eluted between ovalbumim (mol. wt 45 000) and chymotrypsinogen (mol. wt 25 000). Under some experimental conditions, thiodigalactoside, the most potent inhibitor of lectin activity, inhibited the aggregation of cells of the extra-embryonic endoderm of the primitive chick embryo.


Phenylglyoxal is not a selective inhibitor of phagocytosis.

In 1976, it has been reported that phenylglyoxal (C6H5COCHO) selectively inhibits endocytosis in phagocytes of rabbit and mouse. We have tested the specificity of this compound by measuring its effect on human neutrophil chemotaxis, respiration and release of lysosomal enzymes. Pretreatment of human neutrophils with 100 microgram phenylglyoxal/ml for 30 min at 37 degrees C resulted in almost complete inhibition of phagocytosis of opsonized zymosan. However, after treatment with phenylglyoxal, spontaneous mobility as well as chemotaxis of these cells towards casein, rosette formation with opsonized zymosan, stimulation of the oxidative metabolism and release of lysosomal enzymes were also severely decreased. Most of these functions were only partially restored by resuspension of the cells in a medium without phenylglyoxal. The intracellular level of ATP was not affected by phenylglyoxal, but the level of reduced glutathione was decreased. We conclude from the inhibitory action of phenylglyoxal on the stimulated oxygen consumption and its reaction with intracellular glutathione that phenylglyoxal does not necessarily act exclusively on the outside of the plasma membrane. From our studies, it follows that phenylglyoxal is not a specific inhibitor of endo- or exocytosis in human neutrophils. Phenylglyoxal can be used effectively in the bacterial-killing test of phagocytes to inhibit intracellular killing after an initial period of ingestion.


The time course of male meiosis in the red-backed salamander, Plethodon cinereus.

Tritiated-thymidine autoradiography was used to follow the progress of cells through meiosis in male P. cinereus at 20 degrees C. Spermatocytes spend 7 days in leptotene, 5 days in zygotene and 3 days in pachytene before entering the diffuse stage. Diffuse lasts for 8 days and is followed by a diplotene of 2 days. First and second meiotic metaphase occur a total of 26 days after the end of premeiotic S. Considering the information for P. cinereus together with that for 3 other species, it appears that in amphibians the duration of meiosis does not show the linear relation with C-value that has been described for other groups of organisms.


Specific end-to-end attachment of chromosomes in Ornithogalum virens.

C-banding of nonhomologous chromosomes in haploid generative nuclei of Ornithogalum virens (n = 3) reveals a high degree of specificity with respect to end-to-end connexions. The centromeric end of chromosome 2 preferentially associates with the centromeric end of chromosome 3 and the telomeric end of chromosome 3 associates preferentially with the telomeric end of chromosome 1. This same association of nonhomologous chromosomes persists in prophase nuclei of diploid root tips. In addition, the telomeric ends of the 2 chromosome 2s are connected to one another as are the centromeric ends of the chromosome 1s. This results in a ring of chromosomes in which homologues lie opposite one another. Centromeric ends lie on one side of the nucleus and telomeric ends on the other. It is proposed that this specific association of chromosome ends reflects an order which was probably established at the preceding anaphase or telophase and which persists throughout interphase. The suggestion is made that the proximity of homologous ends and consequently homologous alignment may facilitate initiation of pairing at meiosis.


The organization of supercoiled DNA from human chromosomes.

Nucleoids obtained from metaphase-arrested HeLa cells by gentle lysis in non-ionic detergent and 2 M salt were prepared for electron microscopy by Kleinschmidt spreading techniques. From biophysical studies nucleoids have been shown to contain high molecular weight DNA which is supercoiled and topologically constrained. The contents of nucleoids disperse and collapse into 2 dimensions on the water surface as discrete particles (spreads), which are provisionally equated with metaphase chromosomes. In some cases separate spreads are linked. The structure of the spreads is complex. A preliminary description is presented, although we believe that some of the structures reported may be products of preparation and spreading rather than significant features of the organization of intact nucleoids. There is considerable variation in the appearance of different spreads which may be related to the degree of unfolding, spreading or damage of the different preparations. Each spread consists of one or more core areas surrounded by a network of fibres. Cores are composed, at least in part, of compressed fibres and superficially appear to constrain fibres of the network. In the network the inter-twining of pairs of fibres and the occurrence of flat fibre spirals (disks) are interpreted as evidence of DNA supercoiling, but other fibres of similar thickness are not visibly supercoiled. High-order structures including groupings of disks are observed: these may be the structural correlates of the folded domains of supercoiled DNA established biophysically. The relative proportion of disks and other network structures varies between spreads.


Detection of small amounts of human DNA in human-rodent hybrids.

A method of measuring semi-quantitatively small amounts of human DNA in irradiated human x mouse and irradiated human x Chinese-hamster somatic cell hybrids is described. One method uses molecular hybridization of cell DNA bound to nitrocellulose filters with a cRNA probe to Cot 0--1 human DNA. Alternatively hybrid cell DNA is reassociated in solution with a Cot 0--1 fraction of nick-translated human DNA. Formamide buffers give specificity to the reaction. The detection limit of the filter method is 0.2--0.5% equivalents and reassociation kinetics 0.005--0.1% equivalents of a human genome. Experiments with cell hybrids suggest that a fragment of repetitive DNA may be retained along with the selected genes after a cell fusion. In one case however, of a hybrid cell in which malignancy is suppressed, highly repetitious sequences were not found.


Structural changes in the pollinated and unpollinated avocado stigma and style.

Structural changes in the pollinated and unpollinated avocado (Persea americana Mill) stigma and style up to 42 h after first opening of the flower were investigated using light and electron microscopy. The pollen tubes grew in the stigma secretion and intercellular substance and initial contact occurred between the plasma membrane of the male and the cuticle and stigma secretion of the female. The pollen tube wall started to develop 15 min after pollination and increased in thickness up to 24 h after pollination. By 18 h after first opening of the flower, starch had disappeared and cell wall thickenings were present in both the pollinated and unpollinated stigma and style. The wall thickenings developed more slowly in the unpollinated than in the pollinated tissue. They contained lipid and were bounded by callose. Degeneration of the cytoplasm of some of the papilla and transmitting tissue cells occurred only following the passage of the pollen tubes and may be of importance in tube nutrition. There was no degeneration in the unpollinated stigma and style and the cytoplasm did not start to lose clarity until 42 h after first opening of the flower.


Developmental mechanisms in heterospory: cytochemical demonstration of spore-wall enzymes associated with beta-lectins, polysaccharides and lipids in water ferns.

Cytochemical methods are used to examine the distribution and localization of acid phosphatase, non-specific esterase, ribonuclease and peroxidase activity in the walls of the spores of the heterosporous Marsileaceae before and during germination. In the quiescent spore, the principal activity is associated with the perine layer of the wall and the intine, with some activity in the outer, gelatinous wall layer, but none in the exine. The microspores of Marsilea and Pilularia have non-specific esterase activity concentrated in the intine inthe immediate vicinity of the germinal site; that is, above the position of the future male gametangia. The enzymes are not leached from the wall during hydration of the spores, although ribonuclease is redistributed during imbibition with a high concentration of activity remaining in or around the germinal site. The wall enzymes occur together with PAS-reactive and acidic carbohydrates, lipids, and in the microspore perine, beta-lectins. Following the enzyme pattern, the beta-lectins are found to be concentrated in the region of the germinal site. beta-Lectin activity is absent from the megaspore wall. Acidic carbohydrates are confined to the gelatinous wall layer and this layer also binds concanavalin A. In contrast to what has been found for other plant cells, the spore-wall beta-lectins are not water-labile; the activity is not significantly diminished after hydration. This surprising stability suggests that these molecules, together with the enzymes, may be retained in position in the wall by the waterproof overlay of lipid. From the evidence of preliminary developmental studies, it appears that the enzymes associated with the perine layer of the wall originate in the sporophytic tapetal periplasmodium and inclusion of the activity is concurrent with wall differentiation, while the activity associated with the intine is derived from the gametophyte. It is possible, however, in the megaspore at least, that the distribution of the activity may to some extent be influenced by a system of exine channels which communicates between the two domains of the wall during sporogenesis. No definite information is obtained concerning the utility of the enzymes and associated molecules in the life of the spore. Acting separately or in co-operation, their role could conceivably be connected with one or more of four processes; wall differentiation, gametophyte nutrition, gametophyte protection or reproduction. Each of these possibilities is discussed.


Vesicles in guard-cell walls and their possible roles in the stomatal mechanism.

A variety of vesicular inclusions have been observed in guard-cell walls. They have been seen in a number of species and are mostly membrane-bound. They appear confined to the upper and ventral walls of the guard cell. A number of possible origins and functions are discussed. They may be involved in wall deposition or cuticle formation possibly in a role similar to ectodesmata. They also may serve to increase the wall-plasmalemma interface to enhance the movement of ions during the stomatal mechanism.


Ultrastructural localization of calcium-binding sites in the electrocyte of Electrophorus electricus (L.).

Calcium-binding sites were detected in the electrocyte of Electrophorus electricus (L.) using the Oschman & Wall technique, in which CaCl2 was added to the fixative and washing solutions. Deposits were seen scattered along the plasma membrane of the electrocyte, inside mitochondria, associated with the post-synaptic membrane and the membrane of synaptic vesicles.


O-serotyping Providencia alcalifaciens.

The O-serotyping scheme for Providencia was tested on Providencia alcalifaciens isolates collected mostly from two hospitals. The specificites of the somatic (O) antigens of P. alcalifaciens were found to be different from those of Providencia stuartii, and separation of the Providencia typing scheme to allow separate typing of each species led to more efficient typing. All but 4 of 86 isolates were typable. Eighteen serotypes occurred among 53 typable isolates obtained from a pediatric hospital, and 11 occurred among 19 isolates from a general hospital. Thirty-two percent of the isolates from the pediatric hospital belonged to serotype O3, the most frequently isolated and most widely distributed type. The use of the serotyping scheme for P. alcalifaciens is advocated for further studies to examine strains of the species for enteropathogenic types.


Identification of diaminopimelic acid in the Legionnaires disease bacterium.

Diaminopimelic acid was found to be a component of the cell wall of the Legionnaires disease bacterium, thus providing additional evidence that the organism is a bacterium. The presence of this amino acid was determined by gas-liquid chromatography and confirmed by gas chromatography-mass spectrometry.


Serological cross-reactions between Acinetobacter calcoaceticus and chlamydiae.

A cross-reaction between Acinetobacter calcoaceticus and chlamydiae is described. A water-soluble, heat stable, non-dialyzable antigen was extracted from Acinetobacter species by boiling. This antigen fixed complement in the presence of homologous hyperimmune sera from rabbits or guinea pigs and in the presence of heterologous human or hyperimmunized animal sera containing chlamydial antibodies. Hyperimmune antisera to the extracted antigen, or to suspensions of live acinetobacters, also reacted in complement fixation with a group-specific antigen.


Application of O-serotyping in a study of Providencia rettgeri (Proteus rettgeri) isolated from human and nonhuman sources.

A somatic (O) antigen serotyping scheme for Providencia rettgeri (Proteus rettgeri) was modified to exclude O-type strains recently reclassified as urea-positive Providencia stuartii and was extended to include new serotypes to provide for serotyping on the basis of 93 O-antigens. Isolates from two hospitals, five public health laboratories, and nonhuman sources (polluted water and frogs) were serotyped. The 112 isolates collected from a large general hospital over a 99-month period were distributed among 42 O-serotypes. No serotype showed significant predominance that would suggest the occurrence of human strains that are more prone than others to cause human infections, but in an institution experiencing cross-infection, 11 of the 22 (50%) isolates belonged to one serotype. The 54 isolates from the five public health laboratories belonged to 33 serotypes, 15 of which were found also among hospital isolates. All but 5 of 99 frog isolates were typable, and the 94 typable isolates were separated into 25 serotypes. Each of the four isolates from polluted water samples was of a different serotype. Sixteen of the serotypes found in frogs and three found in water were also identified among human isolates.


Effect of storage temperature on viability of Trichophyton mentagrophytes in infected guinea pig skin scales.

The effect of storage temperature on the viability of Trichophyton mentagrophytes in infected guinea pig skin scales was quantitatively evaluated for 182 days. Infected skin scales were stored, while protected from light, in glass containers with desiccant at four temperatures and sampled after saline treatment at various time intervals. No decrease in viability was observed for storage at 4 degrees C, room temperature, or 30 degrees C. Storage at 37 degrees C resulted in a significant loss in viability.


Medium for selective isolation of Fusobacterium nucleatum from human periodontal pockets.

A selective medium, CVE agar, was developed for the isolation of Fusobacterium nucleatum from subgingival plaque of periodontally diseased patients. The medium contained 1.0% Trypticase (BBL Microbiology Systems), 0.5% yeast extract, 0.5% NaCl, 0.2% glucose, 0.02% L-tryptophan, 1.5% agar, and 5% defibrinated whole sheep blood. Erythromycin and crystal violet were added as the selective inhibitory agents at concentrations of 4 and 5 micrograms/ml, respectively. The medium permitted almost total recovery of F. nucleatum when compared with a nonselective medium and suppressed the recovery of most remaining species by nonselective medium and suppressed the recovery of most remaining species by 6 to 8 orders of magnitude. Microorganisms suppressed to a lesser degree included Selenomonas sputigena, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and some strains of Peptostreptococcus. The distinct colonial morphology of F. nucleatum on CVE agar made differentiation relatively easy when contaminants were present. With this medium, F. nucleatum was enumerated from 278 subgingival plaque samples and accounted for less than 1.0 to greater than 25% of the cultivatable microbiota.


Bacteremia associated with Enterobacter sakazakii (yellow, pigmented Enterobacter cloacae).

A case report of bacteremia due to Enterobacter sakazakii, listed previously as yellow-pigmented Enterobacter cloacae (R. Sakazaki, in R. E. Buchanan and N. E. Gibbons, ed., Bergey's Manual of Determinative Bacteriology, 8th ed., p. 325, 1974), occurred in a 7-day-old, Caucasian male who responded successfully to ampicillin therapy. The source of the infection was not known; however, because of the time lapse between birth and the onset of symptoms, the infection was thought to have occurred postnatally.


Time of lymphocyte response after onset of tularemia and after tularemia vaccination.

Blood lymphocytes were prepared from 6 patients at various time intervals after the onset of tularemia and from 10 subjects after vaccination against this disease. Lymphocytes were also prepared from subjects who had been vaccinated 1 and 2 years previously. The lymphocytes were incubated in the presence of membranes of the vaccine strain. Lymphocytes obtained 2 weeks or later after onset of the disease responded to the membranes with increased deoxyribonucleic acid synthesis, whereas lymphocytes obtained earlier than 2 weeks after onset did not respond. Lymphocytes of the vaccinated subjects did not respond to the membranes of the vaccine strain before vaccination. Two to 4 weeks after vaccination lymphocytes from six of the vaccinees yielded a high response, and this response was consistently high for several months. Lymphocytes from four of the vaccinated individuals responded to a low extent only, and this was consistently low for several months. Lymphocytes from individuals vaccinated 1 year before testing responded to a similar extent to the membranes, as did lymphocytes from those who had been vaccinated 1 month previously. Lymphocytes from individuals vaccinated 2 years previously, however, showed a diminished response to the membranes. There was no correlation between titer of agglutinating antibodies and magnitude of lymphocyte reactivity.


Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin.

Stools from patients with antibiotic-associated diarrhea or colitis were cultured to detect the presence of Clostridium difficile. All specimens contained a cytotoxin which was neutralized by Clostridium sordellii antitoxin. Initial testing employed several methods with comparative merits in recovering this organism. These included the use of nonselective media, antibiotic-incorporated media, alcohol shock, and paracresol-containing broth. Optimal results were achieved with primary plating of serial dilutions onto a selective agar containing cycloserine and cefoxitin. This technique was then employed in a large number of specimens. The overall results showed that C. difficile was recovered in specimens from 71 of 73 patients. All isolates of C. difficile produced a cytotoxin which was neutralized by C. sordellii antitoxin in vitro. These results verify the utility of this medium and support the concept that C. difficile accounts for the cytotoxin found in stools in nearly all cases.


Methods of measuring zones of inhibition with the Bauer-Kirby disk susceptibility test.

Standard Bauer-Kirby disk tests were performed with 85 selected isolates, each tested in triplicate by four different investigators. Each disk test was observed, and zone diameters were measured, under two lighting conditions (transmitted light and reflected light). The two lighting systems produced similar zone measurements (+/-2 mm) with 96% of the tests. When there were greater differences, zones appeared to be larger when observed with reflected light. Interlaboratory reproducibility was much greater when using reflected light rather than transmitted light. We concluded that zone diameters should be measured from the back of the plate while it is resting on, or held 2 to 3 inches [ca. 5.1 to 7.6 cm] above, a black, nonreflecting, flat surface, illuminated by a reflected light source.


More sensitive test agar for detection of dextranase-producing oral streptococci and identification of two glucan synthesis-defective dextranase mutants of Streptococcus mutans 6715.

A more sensitive test agar was developed to detect oral microbes with relatively low dextranase activity and to identify dextranase-negative mutants. Several oral streptococci that had previously been scored as dextranase negative readily decolorized the new, blue dextran-containing medium. To assess whether dextranase plays a role in glucan synthesis by oral streptococci, various glucan synthesis-defective mutants were tested for dextranase activity on the new medium. Mutants 4 and 27, which do not cause smooth-surface caries and which synthesize more soluble glucan than their parent, Streptococcus mutans 6715-13, were markedly deficient in these dextranase activity tests.


Lack of correlation between interferon production of mononuclear cells and virus replication in chronic hepatitis B virus infection.

Interferon production in hepatitis B virus carriers with normal liver functions was preserved, whereas in carriers with chronic hepatitis it was depressed. The lower interferon production in hepatitis B virus carriers with chronic hepatitis appears to be the result rather than the cause of chronic hepatic disease.


Cellular fatty acid composition of Francisella tularensis.

Several unusual fatty acids characterized strains of Francisella tularensis. Long-chain (C20-C26) acids and the hydroxy acids 2-hydroxy-decanoate, 3-hydroxy-hexadecanoate, and 3-hydroxy-octadecanoate appeared to be of special diagnostic value.


Enhancement of Ureaplasma urealyticum growth on a differential agar medium (A7B) by a polyamine, putrescine.

Growth of Ureaplasma urealyticum isolates from clinical exudates and urine specimens was significantly improved by supplementation of a Ureaplasma differential agar medium by putrescine. The improved medium was designated medium A7B.


Serum immunoreactive trypsin concentration after a Lundh meal. Its value in the diagnosis of pancreatic disease.

The changes in serum trypsin concentration have been measured in 47 subjects for up to 2 hours after a Lundh meal. In 18 healthy controls, mean fasting trypsin concentration was 285 +/- 125 ng/ml (mean +/- 2 SD). The maximum increase after the Lundh meal (the trypsin response ratio) was 6.7 +/- 7.5%. Six patients with chronic renal failure had elevated fasting serum trypsin concentrations (range 460-1100 ng/ml) but trypsin response ratios fell within the control range. Of five patients with relapsing pancreatitis, two had raised and three normal or low fasting trypsins. After stimulation two had elevated trypsin response ratios; one of the two had evidence of main duct obstruction. Eleven out of 12 patients with chronic pancreatitis (with or without insufficiency) had low fasting trypsin concentrations (range 0-120 ng/ml) Seven of the 12 also had raised trypsin response ratios. In six patients with cancer of the pancreas, fasting trypsin was low in three, normal in two, and raised in one. Both patients with a normal fasting level had a raised trypsin response ratio. The combination of a single estimation of fasting serum trypsin concentration followed by serial measurements after a Lundh meal provides a useful screening test for chronic pancreatic disease.


C3d antiglobulin haemagglutination of human red blood cells. A demonstration of two types of cell-bound C3d by means of typsin digestion.

Washed human red blood cells from blood collected in EDTA were tested by Auto-Analyzer for a percentage of maximum antiglobulin haemagglutination (AH) using monospecific antisera to human C3d and C3c. The cells from normal persons were found to be agglutinated by anti-C3d but not by anti-C3c. To a fixed dilution of antiserum, the normal C3d AH values (X +/- 2 SD) were 34 +/- 19% for adult cells (n = 29) and 14 +/- 19% for cord cells (n = 19); the difference was significant (P less than 0.0001). By pretreatment of these cells with trypsin the C3d AH was either completely abolished or markedly reduced. Its difference between the adult and cord cells was eliminated as the observed values were 4 +/- 7% and 3 +/- 4% respectively (P = 0.15). The supernatant fluid of cell-trypsin mixture, treated with trypsin inhibitors, was found to be inhibitory to C3d AH but not to C3c AH. In contrast, the AH of C3d-coated red blood cells resulting from complement fixation in vivo (ie, cold agglutinin disease) or in vitro (eg, sucrose water reaction) was resistant to trypsin treatment. The difference between the trypsin-sensitive and trypsin-resistant cell-bound C3d is postulated to be at its attachment mechanism to the cell membranes. In addition, both the advantage and limitation of using trypsinised cells for C3d antiglobulin tests are demonstrated.


False-positive antiglobulin tests in healthy subjects and in hospital patients.

An antiglobulin reagent containing anti-IgG, -C4c, -C4d, -C3c, and -C3d was used to test red blood cells from healthy subjects and from patients in hospital. In tests read macroscopically on opal tiles, no positive reactions were found in the healthy subjects. Positive reactions were, however, observed in 7% of the hospital patients. Further tests with monospecific reagents showed the positive reactions to be due to the presence of C4d and C3d on the red cells. All patients with positive antiglobulin reactions had serious disease, in most cases associated with abnormal antibody production or abnormal immunoglobulin levels.


Use of low ionic strength saline for crossmatching and antibody screening.

A totally revised crossmatching and antibody screening procedure has been designed using low ionic strength saline as the suspending medium for red cells in the saline and enzyme phases as well as the antiglobulin test. The replacement of Pasteur pipettes with precision samplers for all serum and red cell dispensing has resulted in improvement in the reproducibility and standardisation of technique. The major advantages are improved sensitivity, shortened incubation time, and simplicity of technical performance.


Splenomegaly and splenectomy in sarcoidosis.

The natural history of 30 patients with sarcoidosis who showed histological evidence of granulomatous involvement of the spleen has been studied; 24 patients had splenomegaly, 16 of whom had splenectomy. The main indication for splenectomy was splenomegaly and resultant discomfort. Corticosteroids reduced spleen size but reduction or withdrawal of the relatively high dosage required resulted in rebound splenomegaly within a period of three months to three years. Haematological abnormalities were controlled by splenectomy in all patients so affected, but the natural history of their sarcoidosis remained unaltered.


Clinical evaluation of eosinophils in the sputum.

The sputum differential eosinophil/neutrophil count was done in 384 patients using Leishman staining. The patients were distributed in four groups: bronchial asthma (197 patients); chronic bronchitis with wheezing (45 patients); chronic bronchitis and/or emphysema without wheezing (73 patients); other pulmonary diseases (64 patients). Eosinophils were present in patients from all groups but more frequently (P less than 0.001) in asthma: 142 (72%) of 197 patients. In bronchial asthma and chronic bronchitis with wheezing the percentages of eosinophils were more frequently (P less than 0.001) above 80%: 57% and 58% of the patients respectively. The other two groups had more cases with 19% or less eosinophils. There is no percentage level specific for asthma but levels above 80% of eosinophils are strongly suggestive of asthma or of chronic bronchitis with wheezing.


Analysis of reproducibility of subjective grading systems for breast carcinoma.

The inter- and intra-observer reproducibility of three grading systems for breast cancer has been analysed: those of the World Health Organisation, of Black, and of Hartveit. In addition, the components forming the basis of these grading systems were analysed separately with regard to reproducibility and interrelationship. In this analysis, degree of differentiation was also included as a parameter. The grading systems of WHO and of Black gave a stratification of the material into three categories of tumours, but that of Hartveit did not. All three systems had a low inter- and intra-observer reproducibility. Truncated component analysis indicated that the grading systems of WHO and of Black are closely related to each other and to the 'nuclear lobulation' component of Hartveit's system. The components 'pleomorphism', 'mitotic frequency', and 'hyperchromasia' of the WHO system were closely interrelated. Descriptors such as 'differentiation' and 'tubule formation' were interrelated but acted in a different direction from the others.


Campylobacter colitis.

A patient is described in whom a campylobacter enteritis closely resembled ulcerative colitis on clinical, sigmoidoscopic, and histological grounds. Selective stool culture techniques may be necessary to differentiate campylobacter colitis from ulcerative colitis proper.


Acute diarrhoea: Campylobacter colitis and the role of rectal biopsy.

Campylobacter spp. were the organisms isolated most frequently from 29 consecutive patients admitted with acute diarrhoea to an infectious disease unit. Rectal biopsies taken from 21 of the patients were abnormal in all but four, and in the patients with campylobacter infection there was a characteristic proctocolitis in each case. The histopathology was similar to that described for salmonella and shigella infections but clearly different from typical ulcerative colitis and Crohn's disease. Therefore in patients with acute diarrhoea we suggest that selective culture for Campylobacter spp. should be made a routine and that rectal biopsy has an important diagnostic role, particularly in patients with negative cultures.


Prevalence of voice quality deviations in the normal adult population.

The purpose of this study was to determine the prevalence of voice quality deviations in a normal adult population. One-hundred twelve subjects, aged 17 to 80, read a short paragraph aloud into a high-fidelity tape recorder and completed a case history questionnaire. A group of 11 pretrained judges rated overall performance of each taped sample on a seven-point equal-appearing intervals scale, then designated those quality components which contributed toward deviant ratings. Eighty-two percent of the group received a mean severity rating lower than 1.99; 16% had a rating between 2.00 and 2.99; and 2% was assigned a mean rating higher than 3.00.


Establishing functional language in an autistic child: a cooperative approach.

The case study of a 7-yr-old boy who was diagnosed as autistic is reported. The cooperative efforts of a teacher of the severely emotionally disturbed, a speech pathologist, and the child's parents were combined to create an optimal situation for the facilitation of behavior change and language development. Examples of the procedures used and the child's progress over a 2 1/2-yr period are presented and described.


Effect of prestimulation on sentence comprehension by aphasic subjects.

Aphasic subjects and controls were tested to determine whether comprehension of spoken sentences was enhanced by prestimulation concerning their contents. For each of the 20 test points, the subject listened to the test sentence and selected the corresponding picture from among four pictures presented simultaneously. The preparatory suggestions were given in one test condition and withheld in the other. Each prestimulation consisted of one or two sentences that indicated the persons, objects, or general situation to be presented in the test sentence. Analysis of variance indicated that the effect of the prestimulation was not significant.


Pragmatics and elicited imitation: children's performance on discursively related and discursively unrelated sentences.

Two experiments were conducted to determine the elicited imitation performance of preschool children when the stimulus sentences were meaningfully related to each other in a story as compared to when the sentences were arranged in a random order. Experiment 1 used a repeated-measures design and found a practice effect demonstrating less errors during the second trial regardless of experimental condition. Experiment 2 implemented a group design with one group imitating the related (story) sentences and the other imitating the randomly arranged sentences. The results showed no significant difference in the mean number of errors occurring in the "story" versus the "random" condition. The implications of the practice effect found in Experiment 1 are discussed and the error pattern during the second trial is evaluated. The efficacy of using discursively related as opposed to unrelated sentences in elicited imitation tasks is discussed from a pragmatic point of view.


Markedness analysis of phonemic substitution errors in apraxia of speech.

The phonemic substitution errors of ten apractic speakers were analyzed to investigate the effects of markedness on speech production patterns. Results revealed that: more errors were produced on phonemes high in markedness; more changes in markedness were from marked to unmarked than unmarked to marked; and errors and directional changes in markedness were positively correlated with ease of articulation. The findings were interpreted as a systematic effort by the apractic speakers to reduce the complexity of articulatory gestures required to produce phonemes.


The emergence, localization and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. I. Gamma aminobutyric acid.

The high-affinity uptake, biosynthesis and release of GABA have been studied in the retina of Xenopus laevis. In the mature retina, [3H]-GABA is accumulated predominantly by horizontal cells. A second population of cells located in the inner nuclear layer (possibly a type of amacrine cell) also showed a specific GABA uptake. In addition, this retina contains significant activities of L-glutamic acid decarboxylase and also releases [3H]-GABA in response to increasing K+ concentrations in the medium. We have followed the appearance and maturation of these GABA-ergic properties during embryonic development of this retina. Our results indicate that these properties emerge in a precise temporal pattern during retinal differentiation: the specific neuronal uptake of GABA precedes GABA synthesis which is followed by K+-stimulated GABA release.


Microcircuitry of cat visual cortex: classification of neurons in layer IV of area 17, and identification of the patterns of lateral geniculate input.

Neurons in the cerebral cortex have been classified primarily by their differences in axonal and dendritic branching patterns observed in material impregnated by the Golgi method. Although these morphological differences are widely believed to reflect differences in connectivity, very little is actually known about the patterns of synaptic input to different cell types. We have obtained such information for 32 adjacent neurons in layer IVab of cat cortical area 17 by reconstructing them from electron micrographs of 150 serial sections. Synaptic terminals from the lateral geniculate nucleus were labeled in this material by anterograde degeneration and their distribution, as well as that of normal terminals containing flat or round vesicles, was recorded. The neurons were divided into seven classes based on differences in size, shape, dendritic branching pattern and synaptic input. Class I cells were pyramidal with apical and basilar dendrites, dendritic spines, exclusively flat-vesicle terminals on the somas (11/100 micron2), and geniculate terminals  on the basilar dendrites. Class II cells were large stellates (20 micron diameter) with dark cytoplasm and numerous flat-vesicle and round-vesicle terminals on the somas (48/100 micron2). Geniculate terminals contacted the cell bodies and primary, secondary, and tertiary dendrites. The Class III cell was stellate with varicose dendrites, a sparse distribution of flat-vesicle terminals (8/100 micron2) on the soma, and both geniculate and round-vesicle terminals on the dendrites. Class IV cells had radially elongated somas with sharply tapered apical and basilar dendrites bearing spines. There was a medium distribution of flat-vesicles terminals (17/100 mu2), to the somas while geniculate terminals were restricted to the secondary dendrites. Class V cells were multipolar with flat-vesicle terminals on the somas (11/100 micron2) and a few geniculate terminals on the dendrites. Class VI cells were mostly small (as small as 7 micron diameter), with a sparse distribution on the somas of both flat-vesicle terminals (7/100 micron2). Two cells had geniculate terminals on their somas. Class VII cells had sharply tapered apical and basilar dendrites, both flat-vesicle and round-vesicle terminals on the somas (14/100 micron2), and no geniculate input. The results make clear that the neurons in layer IVab are quite heterogeneous, not merely in their intrinsic morphology, but also in their patterns of connectivity. The geniculate input is not funneled to a single type of neuron but diverges widely, contacting at least six different cell types, and may form on each a pattern that is characteristic for the type. The reconstruction approach, in providing a detailed identification of the synaptic patterns on substantial numbers of adjacent cells, should make it possible to address directly certain unanswered questions about cortical circuitry...


Relations between cell body size, axon diameter and axon conduction velocity of triceps surae alpha montoneurons during the postnatal development in the cat.

Triceps surae alpha-motoneurons in cats of different postnatal ages were stained intracellularly with horseradish peroxidase (HRP) and studied light microscopically. In individual neurons, the mean diameter of the cell body and the intramedullary axon diameter were measured and related to the axon conduction velocity. The mean diameter of the cell body grew from 39.6 micrometer at birth to 57.6 micrometer in the adult cat, while the corresponding figures for the intramedullary axon diameters were 2.4 micrometer and 6.7 micrometer. During the same period of time, the axon conduction velocity increased from 11.3 m/s to 93.5 m/s, and the ratio between the conduction velocity and the intramedullary diameter of the axon (CV/d ratio) increased from 4.6 to 14.1. The results indicate that the growth of the cell body is smaller and completed earlier than the growth in diameter of the intramedullary and, in particular, the peripheral parts of the axon. The considerable change of the CV/d ratio during the postnatal development may be explained by previously described immature morphological properties of the axons in very kittens, and by a changing relation between the dimensions of the intramedullary and peripheral parts of the axon.


Melanoma mimicking plantar wart.

A 58-year-old white man had an enlarging lesion on the sole of the right foot mistakenly diagnosed clinically and histologically as verruca vulgaris. Lack of response to therapy led to a repeat biopsy which showed malignant melanoma, acral lentiginous type, level IV. The lesion was surgically removed, and 11 months later there was no evidence of recurrence. In the differential diagnosis of lesions of the palms and soles, one should include acral lentiginous melanoma, especially if the lesion is pigmented.