Nutritional Biochemicals Corporation Research Articles

Electrochemical Studies of Amino Acid: Metal Ion Interactions

Introduction Previous studies (1-3) in this laboratory have involved electrochemical studies of the interaction of Zn(II) ions with L-cysteine in an attempt to more fully characterize the formation of “zinc finger” proteins (4). Subsequent work has involved similar investigations of L-histidine (5), and the work has now been extended to L-tryptophan. Clear evidence for the formation of Zn(II) complexes with L-cysteine and L-histidine has been obtained by cyclic voltammetric peak potential shifts for zinc ion reduction (2, 5). In the present study, we have extended this work to L-tryptophan by means of voltammetric studies of the amino acid and by investigation of its interactions with metals such as zinc and copper. Results for the interaction of bismuth(III) with L-cysteine and with glutathione, prompted by the significance of such interactions in human biochemistry (6-8), are also planned for presentation. Experimental L-Cysteine, L-glutathione, L-histidine, and MOPS (3-(N-morpholino)propanesulfonic acid) were obtained from Sigma-Aldrich Corporation, and L-tryptophan was obtained from Nutritional Biochemicals Corporation. Electrochemical experiments were carried out under nitrogen using a Gamry Instruments Interface 1000 potentiostat and Gamry Framework software. Working electrodes were obtained from BASi (Glassy carbon, 3.0 mm diameter; platinum 1.6 mm) and eDAQ (gold, 1.0 mm diameter). Potentials were measured with respect to a silver/silver chloride saturated KCl reference electrode (BASi). Results and Discussion Electrochemical studies of L-tryptophan in pH 7.4 phosphate buffer have shown that this amino acid undergoes oxidation at +0.82 V vs Ag/AgCl at 100 mV/s, which is a considerably less positive potential than that observed for L-histidine (5). The effects of L-tryptophan additions to ZnSO4 added to pH 7.4 MOPS buffer were found to be generally similar to those for L-histidine (5). For the case of interactions of L-cysteine with Bi(III) in pH 7.4 MOPS buffer, Bi(NO3)3 was added to pH 7.4 MOPS buffer resulting in the formation of slightly soluble hydroxyl bismuth complexes. Upon addition of 1:1 bismuth(III):L-cysteine, the solution appearance changed from cloudy to clear, and voltammetric currents increased substantially, indicating a strong interaction between bismuth(III) and L-cysteine. In addition, a negative voltammetric shift for the bismuth(III) reduction potential was observed, as was the case for the zinc(II):L-cysteine interaction (2). Further additions of L-cysteine were found to produce additional negative shifts for bismuth(III) reduction. Similar behavior for L-glutathione was observed. MALDI-TOF spectra for the 1:2 Bi(III):L-glutathione and similar complexes in aqueous solutions have been reported (10) and provide supporting evidence for the electrochemical results in the present study. A similar spectrum for the 1:2 Bi(III):L-glutathione complex in pH 7.4 MOPS buffer has been obtained in the present work.

Developmental Changes in the Pattern of Lactate Dehydrogenase Isoenzymes of Rat Submandibular Gland

With the aid of electrophoretic techniques it was shown that lactate dehydrogenase (LDH, EC. 1.1.1.27) of animal tissues and body fluids consisted of at least five isoenzymes. The properties of LDH isoenzymes of rat submandibular gland were reported [1]. In the present report, changes of LDH isoenzyme activities of submandibular gland were investigated during development in the rat. Reduced nicotinamide-adenine dinucleotide (reduced NAD) was obtained from Boehringer Mannheim. Bovine albumin was obtained from Nutritional Biochemicals Corporation. Other materials used were also available commercially. Rats of Donryu strain were used. The animals were sacrificed by sharp blow on the head, and their submandibular glands were excised and placed in ice-cold 0.25 M sucrose solution. The tisses were homogenized with tephron homogenizer in 4 volumes of ice-cold 0.05 M barbital buffer (pH 8.6), and the supernatant fraction was obtained after centrifugation at 100, 000 × g for 1 hour. Electrophoretic separation of the isoenzymes was made with starch in a horizontal position. The supernatant fluid obtained from the homogenate was inserted into a slit in the starch with a supporting medium of starch granules. The separation was made in starch block of 30 × 1 × 1 cm. All of the runs were made for 21 hours at a field strength of 4 volts per 1 cm at 5°C. After 21 hours of electrophoresis, the starch was cut into 0.5 cm strips at right angles to the direction. These strips were extracted with 2.0 ml of 0.1 M phosphate buffer (pH 7.4) during 3 hours with occasional stirring, and the clear supernatant fluids were obtained by centrifugation at 800 x g for 10 minutes. The residues were extracted once more with 2.0 ml of same buffer during 1 hour and the supernatant fluids were added to the first extracts. The LDH activity of the supernatant fluid combined in each tube was determined. The enzyme was incubated with 0.33 μmole of reduced NAD, 2.3 μmoles of sodium pyruvate in phosphate buffer (680 pmoles, pH 7.4) in the final volume of 2.0 ml for 4 minutes at 25°C. The reduced NAD should be added last of all. Subsequently, the enzyme activity was determined by measuring the rate of optical density decrease at a wave length of 340 mμresulting from the oxidation of reduced NAD. One unit of LDH activity was defined as the amount of the enzyme that decreases 1.0 μmoles of reduced NAD per minute at 25°C. LDH isoenzymes were designated according to PLAGEMANN [2]. Total LDH activity of submandibular gland (expressed on a tissue weight basis) increased steadily throughout the early postnatal period, and thereafter it remained relatively constant throughout the 150-day experimental period and then decreased significantly by the postnatal day 200 (Table 1).The data in Table 1 reveal that LDH isoenzyme patterns of submandibular gland were changed during development in the rat. Especially, LDH5 activity was decreased for the first 10 days after birth, then increased. However, LDH4 activity was increased for the 10 days after birth, then decreased. No activity of LDH1 and LDH2 was found immediately before birth, and no activity of LDH1, LDH2, LDH3 was also found in 120-200-day-old animals. To clarify the physiological significance of developmental changes in the pattern of LDH isoenzymes of rat submandibular gland, further investigations are undergoing in our laboratory.

The detection of relaxin in porcine, ovine and human plasma by radioimmunoassay.

A radioimmunoassay for porcine relaxin is described based on the inhibition of the reaction between I-labeled porcine relaxin and rabbit antiserum to porcine relaxin. The time course of the binding of labeled porcine relaxin to antiserum was determined by chromatoelectrophoresis. The sensitivity of the assay is greater than 4.0 ng of relaxin/ml of plasma. Immunoactivity in plasma from a late pregnant sheep and pig was identical to the standard porcine relaxin. Inhibitions obtained with plasmas from an early pregnant sheep, a ram, boar and normal man differed slightly from that of the standard porcine relaxin. On the other hand, definite partial crossreactions were shown by both pig and sheep plasma after ovariectomy and no inhibition was observed by plasma from a sheep and human after both ovariectomy and hysterectomy. {Endocrinology 91: 1113, 1972) T N 1926, Hisaw (1) described a peptide hor•*mone isolated from the corpora lutea of sow ovaries which produced a marked relaxation of the pelvic ligaments in guinea pigs. Bioassays were subsequently developed based upon the biological activities of relaxin, Hall (2). Current bioassay methods are impractical for measurements of relaxin in multiple samples taken in a wide variety of physiological conditions. Hemagglutination-inhibition has been used for relaxin measurements in saline extracts of ovaries from several species and for cross-reactivity studies by Cohen (3) and McClintock and Zarrow (4). The application of this technique to the estimation of plasma relaxin activity has not been reported. The relaxin preparation used for this study (NIH-R-P1) was extracted from the ovaries of pregnant sows and therefore represents ovarian relaxin as produced during pregnancy. The Uterine Relaxing Factor (URF) content of this preparation is not known. URF has not yet been identified as a separate moiety from relaxin, Griss, Keck, Englehorn and Tuppy (5), isolated from pig ovaries. However, in addition to the possible heterogeneity of ovarian relaxin, plasma may also contain relaxins produced by the decidua, endometrium and placenta, according to Dallenbach and Dallenbach-Hellweg (6). Received January 4, 1972. Address for correspondence: Dr. Gillian D. Bryant, Department of Anatomy & Reproductive Biology, 1960 East-West Road, Honolulu, Hawaii 96822. A sensitive and precise radioimmunoassay has been developed for ovarian relaxin and preliminary results have been obtained in plasma from pig, sheep and man. Evidence of a uterine source of relaxin is shown and discussed. Materials and Methods Buffers. Sodium salts were used in the preparation of buffers. Diluent buffer refers to a solution 0.5 mg/ml of bovine gamma globulin, Fraction II (Nutritional Biochemicals Corporation) in 0.05M-barbitone buffer pH 8.6 with 0.5 mg/ml iodoacetamide (Nutritional Biochemicals Corporation). Horse serum was obtained in liquid form from Grand Island Biological Company. Porcine relaxin. A preparation of porcine relaxin (NIH-R-Pl) was used as a standard and for the preparation of I-labeled porcine relaxin. Polyacrylamide gel electrophoresis at pH 7.6 and 9.6 showed a single band but further studies on the physicochemical homogeneity are in progress. i'-labeled porcine relaxin. This was prepared weekly by the method of Hunter and Greenwood (7) except that 0.5 mCi obtained from Cambridge Nuclear, Mass., U.S.A. was reacted with the hormone (10 Mg in 0.02 ml) and 30 M-g of chloramine-T. The 1 3 Ilabeled porcine relaxin was separated from the unreacted iodide on Sephadex G-25 (1.5 g) previously equilibrated with 1 ml of 0.05M-barbitone buffer, pH 8.6, containing 20 mg of bovine gamma globulin. Eluates (15 X 0.75 ml) in the 0.05M-barbitone buffer were collected into tubes containing 0.1 ml of diluent. The fraction containing the highest number of counts in the protein peak was repurified by passage through Sephadex G-50 (2.5 g) previously

Hymenolepis microstoma Cysticercoid Activation and Excystation In vitro (Cestoda)

Excystation was studied in vitro and the events of excystation in this species were histologically determined and compared with previous reports for Hymenolepis diminuta. Occlusion of the anterior canal during early cysticercoid development in H. microstoma necessitates a method of excystation different from that of H. diminuta in which the anterior canal remains open and provides egress for the larval scolex. Supplementary studies of the qualitative and quantitative effects of various artificial digestive solutions on H. microstoma activation and excystation are included. A possible protective function of the cysticercoid wall during passage through the mouse stomach is discussed in conjunction with histological studies of the priming effect of artificial gastric juice. Natural infections of Hymenolepis microstoma (Dujardin, 1845) occur when mice eat arthropods harboring infective cysticercoids. Activation of the larval scolex and excystation occur in the duodenum subsequent to gastric dissolution of outer cysticercoid membranes (Dvorak, Jones, and Kuhlman, 1961). The adult tapeworm becomes established in the bile duct. Consequently, studies concerning the effects of physicochemical conditions of the vertebrate alimentary canal on cysticercoids contribute to an understanding of both cysticercoid wall function and host specificity. Rothman (1959) employed in vitro techniques to resolve responses of cyclophyllidean larvae to artificial digestive juices. Goodchild and Harrison (1961) studied histologically the excystation of Hymenolepis diminuta cysticercoids and reported that the scolex emerged via a patent anterior canal. We have examined excystation of Hymenolepis microstoma cysticercoids in vitro and have investigated the histology of excystation. In this species, the anterior canal of the cysticercoid is occluded during early development, and therefore it requires a method of excystation different from H. diminuta. MATERIALS AND METHODS Hymenolepis microstoma adults were maintained in inbred C3H/Crgl mice, and cysticercoids were established in adult Tribolium confusum by exposure for 72 hr to macerated, gravid proglottids. Before feeding, proglottids were stored overnight in 0.85% NaCI at 4 C (Collings and Hutchins, Received for publication 7 December 1971. 735 1965). Flour beetles were starved for 10 days prior to exposure and subsequently maintained on rolled oats at 22 to 25 C until used. Depending upon the particular experiment, cysticercoids ranging in age from 25 to 50 days were recovered by dissection in 0.85% saline. Artificial digestive media, prepared immediately prior to use, consisted of 0.5% (w/v) solutions of pepsin (Merck and Co., Inc.), trypsin (Fisher Scientific Company), sodium taurocholate (Nutritional Biochemicals Corporation), or trypsinsodium taurocholate combined in 0.85% NaCl. The pH of these solutions was adjusted with HC1 or NaHCO3 to the desired value. For quantitative studies of excystation, larvae were incubated in various digestive media at 37 C, and counts of excystation events were made using a dissecting microscope with a stage temperature of 37 C. Larvae were considered excysted when the entire scolex was free of the cyst wall. Detailed observations of excystation in living material were made with a compound microscope equipped with a stage incubator (Lab-Line; C. S. and E. Incu-Stage) adjusted to 37 C. Cysticercoids for histological study were incubated in the various digestive fluids at 23 C and were ultimately fixed in Zenker's fluid and embedded in paraffin. Specimens were serially sectioned at 5 Au and stained with Harris's hematoxylin and Triosin or with Mallory's triple stain (Galigher and Kozloff, 1964) for selective staining of fibrous elements (Voge, 1963).

Gallstones induced by normal foodstuffs in dogs.

EXPERIMENTAL models of gallstone formation have been described in many species1,2, but the lithogenic methods used were unphysiological or unnatural. In dogs, spontaneous gallstones are rare1, and human cholesterol gallstones placed in the dog gallbladder will dissolve3. Canine gallstones occur in biliary fistulas4, but the only report of gallbladder stones followed combined cholesterol-feeding and occlusion of the cystic duct5. To induce stones physiologically in this resistant species, we designed a diet in 1965 which hypothetically would alter bile salt–phospholipid–cholesterol–pigment relationships in bile and cause bile stasis as follows: low amounts of protein, high content of sugar and no added bile salts to reduce total bile salt and volume-flow of bile6,7; low content of total and unsaturated fat to reduce and alter phospho-lipids and bile salts in bile and to decrease gallbladder contractions8–10; and added cholesterol to overcome the cholesterol-holding capacity of dog bile1,11. Cornstarch and multiple small feedings were added further to simulate diets of certain human populations with a high incidence of cholelithiasis, for example, the Pima Indians12, or produce biliary stasis1,8. The diet was compounded of purified casein 10 per cent, sucrose 50 per cent, cornstarch 26 per cent, animal lard 5 per cent, cholesterol USP 1 per cent, salt mixture USP XIV 5 per cent, ‘Vitamin Diet Fortification Mixture’ 2 per cent and cellulose as filler 1 per cent (Nutritional Biochemicals Corporation, Cleveland, Ohio).

EFFECT OF SHORT-TERM VITAMIN A DEFICIENCY ON TESTICULAR LIPIDS IN THE RAT

In 1933, Mason reported that long-term vitamin A deficiency caused sloughing of seminiferous tubular elements of the rat testis. Since that time, several workers have reported testicular lesions caused by deficiency of the vitamin (Beaver, 1961; Coward, McHowell, Pitt & Thompson, 1966; Gamball, 1966). Gamball (1966) found changes in testicular phospholipid associated with sterility following vitamin A deficiency. Since other treatments which result in sterility also cause lipid changes in the testis (Fleeger, Bishop, Gomes & VanDemark, 1968; Johnson, VanDemark, Gomes, Butler & Hodgen, 1967), it is possible that testicular lipids reflect the status of the testis, even during early stages of degeneration following deleterious treatments. The present study was undertaken to determine whether testicular lipid levels are affected by short-term vitamin A deficiency and, if so, the nature of such changes. Male Wistar rats, 30 days of age, and weighing about 100 g, were fed a commercially available (Nutritional Biochemicals Corporation, Cleveland, Ohio 44128) rat diet, purported to be vitamin A free, for 5 weeks. Control animals were supplemented with 12 i.u. retinol/g of feed to meet the vitamin A requirement. A body-weight plateau, considered the first sign of deficiency,

Autoradiographic Studies on DNA Biosynthesis in Entamoeba histolytica in CLG Medium

Incorporation of tritium from thymidine-methyl-H3 (TMH3) in the Laredo strain of Entamoeba histolytica grown at room temperature in the CLG medium, as in the K9 strain grown at 37 C, occurs in both nucleus and cytoplasm. Most extensive cytoplasmic activity is detected during periods of most pronounced growth. Nuclear activity is generally evenly dispersed, but in some instances it is restricted to portions of the peripheral chromatin. The cytoplasmic label is shown to be due in part or wholly to ingestion of DNA of the nonmultiplying protoplasts of the associate cell (Bacteroides) used to support growth of amebae in CLG medium. This cytoplasmic label is unstable and at least some of it serves as precursor material for nuclear DNA of amebae. Experiments done with appropriate RNase and DNAse digestions, and unlabeled carrier compounds thymidine, uridine, thymidylic acid and uridylic acid, indicate that TMH' is highly specific for DNA in this cell system and that the thymidine kinase salvage pathway is probably involved in TMH: utilization. The efficacy of studying patterns of DNA biosynthesis in Entamoeba histolytica by autoradiography using H3-thymidine (H3T) has recently been reported (Albach, Shaffer, and Watson, 1966). These studies described the extent of H3T utilization over the growth cycle of the K9 strain of E. histolytica grown at 37 C in the CLG medium with penicillin-inhibited Bacteroides as the associate organism. The uptake of tritium from H3T was found in a DNasesensitive molecule in both nucleus and cytoplasm of the amebae. Most of the cytoplasmic activity, which was very extensive during periods of pronounced growth, was found to be relatively unstable. It was suggested that the source of DNase-sensitive cytoplasmic activity may be partly or entirely due to ingestion of prelabeled bacterial DNA since tritium from H3-thymidine was also incorporated into DNasesensitive material by the nonmultiplying associate organism. It was thus considered possible that the nuclear activity might be derived from the cytoplasmic label. The purpose of this communication is to extend the observations on the utilization of H3T to the Laredo strain of E. histolytica grown at room temperature in the CLG medium. A preliminary report of these observations has been published (Albach and Shaffer, 1966). Further studies done with the K9 strain, which will provide additional evidence to support the hypothesis that much of the cytoplasmic activity is Received for publication 21 July 1967. * Supported by Grant AI-08004-01 from the NIAID. apparently derived from ingested bacterial DNA which is then used for nuclear DNA synthesis will also be included. MATERIALS AND METHODS Entamoeba histolytica was propagated routinely in the CLG (cysteine-lactalbumin-glucose) medium with the penicillin-inhibited Bacteroides sp. as the associate organism (McDade and Shaffer, 1959). Two strains were used, the K9 strain which was incubated at 37 C and transferred three times weekly, and the Laredo strain which was grown at room temperature and transferred once weekly. Incubation of amebae with exogenous thymidine-methyl-H3 (TMH3) and with unlabeled Bacteroides sp. Cultures of E. histolytica were incubated for varying periods of time with various amounts of TMH3 (Schwarz BioResearch, sp. act. 6 C/mM). In some experiments, unlabeled carrier compounds, thymidine (TdR), thymidylic acid (TMP), uridine (UR), uridylic acid (UMP), obtained from Nutritional Biochemicals Corporation, were added to the medium to yield a final concentration of 0.5 mg/ml. This concentration (ca. 2mM) was approximately 500 to 1,000 times that of the labeled nucleoside. Since it has been demonstrated that thymidine at concentrations above 0.02 mxt may inhibit DNA synthesis and cell growth in some cells (Whittle, 1966; Yang et al., 1966), preliminary experiments were done with all carrier compounds to assure that, at the concentration range used, no such inhibition of cell proliferation occurred. The growth responses of amebae propagated with or without carrier compounds were essentially the same. After the prescribed incubation period in the radioactive medium, the amebae were harvested and washed free of the labeled medium and autoradiographs prepared immediately, or the cells were resuspended in unlabeled medium for a predetermined period of time and then autoradiographs prepared.

Effect of Gibberellic Acid on the Senescence of Leaf Discs of Nasturtium (Tropaeolum majus)

Aging in leaves is characterized by a decrease in chlorophyll content and an accompanying loss of protein and ribonucleic acid. It is well known that these symptoms of senescence occur at an accelerated rate in excised mature leaves and in leaf discs floated on water. In recent years it has been established that several chemical substances retard the senescence of leaves. Among the compounds which retard senescence are the kinetins and related structures (4, 6,8, 10, 12) and auixins (3, 9). It is significant that the retardation of senescence by these substances varies from species to species. Thtus, atuxins delay senescence in Prunus leaves but are without effect on Xanthium leaves. In contrast kinetin retards leaf senescence in Xanthium btut is ineffective in Prunus (5). Recently it has been reported (2) that the gibberellins can retard leaf senescence. However, the results are again species specific. Thtus in 17 species stutdied, only Taraxacumt officiwiiile demonstrated a gibberellic acid regulation of senescence (2). During investigations in this laboratory into the senescence of leaf discs of Tropaeolunt majts it was noted that senescence coutld be prevented by gibberellic acid treatment. Thus the Nasturtiutm must be added to the species in which senescence is regutlated by gibberellin. Althouigh the mode of action of gibberellic acid in controlling senescence remains to be eluicidated, the response of leaf discs to gibberellin treatment has been established and is reported here. Nasturtiumll seed 7Tropaeolumw mnajuts (Glorious Gleam) were sown in a vermicuilite: p.at: soil mixtture (1: 1: 1) and germinated and grown in the greenhouse with a minimum temperature of 18? and natuiral illumination. Leaves of approximately 6 cm diameter were collected from these seedlings at intervals as requiired. One-cm discs were punched from the leaves, and 30 discs were floated in 20 ml of either water or gibberellic acid soluition in petri dishes. The gibberellic acid (GA) uised was the 75 % K salt stupplied by Nutritional Biochemicals Corporation and for most investigations the concentration used was 20 mg per liter. The petri dishes and contents were maintained at 220 with an illumination of 600 ft-c during a 12-hour photoperiod. At intervals 10 discs were removed from the dishes and used for chemical analyses. The leaf discs were extracted 3 times with boiling 80 % (v/v) ethanol and then grouind in 80 % (v/v) ethanol in a ground glass homogenizer. The homogenate was centriftuged at 12,000 X g for 15 minutes. The resulting supernatant fluid was combined with the initial extracts and after appropriate dilution used for the estimation of chlorophyll, alcohol-soluble carbohvdrate and alcohol-soluble nitrogen. Chlorophyll was estimated by determining the OD of the alcohol extract at 665 myu (4). Carbohydrate content of the alcohol extract was measured by the anthrone method of Yemm and Willis (13) and the alcohol-soluble nitrogen was determined by a modified Kjeldahl digestion and nesslerization ( 11). RNA, DNA and protein were extracted from the alcohol-insoluble residue according to the methods described by Osborne (4). RNA and DNA were estimated by referring OD readings of the extracts at 260 mu to similarly treated preparations of purified RNA and DNA. Protein content of the alkaline extracts was determined bv a modified Kjeldahl digestion followed by nesslerization (11). Duiring the 7-day period of study the leaf discs floating on water became yellow while those floating on GA solutions remained green. It has been found that GA was effective in preventing yellowing and chlorophyll loss in Nasturtium leaf discs at the lowest concentrations tested, 1 mg per liter. However, as a matter of routine 20 mg per liter solutions were used in all subsequtent experiments. It has been fotund that leaf discs floated in GA solutions of this concentration maintained the&r green color for at least 6 weeks. In contrast leaf discs floated on water turned yellow in 4 to 5 days. These visible differences between control and GA treated leaf discs are confirmed by data oIn chlorophyll content of the alcoholic extracts of the leaf discs. It was foulnd (table I) that over a 7-day period the chlorophyll content decreased 1 Supported by NSF grant GB-2304 and by federal funds granted to the University of Illinois Agricultuiral Experiment Station.

A Comparison of Methods for Quantitative Estimation of Hypoxanthine, Xanthine, and Uric Acid in Insect Material

The purine uric acid is the usual nitrogenous metabolite of protein catabolism in insects. Hypoxanthine and xanthine are purines which are normally converted into uric acid and therefore do not appear in insect exereta; however, since the discovery of all three of these purines in excreta of the sheep ked, Melophagus ovinus Meig., (Nelson 1958) various workers have found them in exereta of three additional species. There is no quantitative information available at present on excretion of hypoxanthine and xanthine in any species. Excretory hypoxanthine and xanthine may be detrimental to insects by interfering with the normal water reabsorption process occurring in the rectum. The possibility of some adverse effect has been suggested by Mitchell et. al. (1959), who showed that there is about fifty per cent failure to successfully emerge from the pupal stage at 25?C by a Drosop,hila melanogaster Meig. mutant, rosy,2 which excretes hypoxanthine instead of uric acid. Both hypoxanthine and xanthine are approximately 25 times as soluble as uric acid (Dawson et. al. 1959), which means that more water must be available for excretion, or that more energy must be expended in reabsorbing needed water against an increased osmotic gradient when these two purines make up a significant amount of the nitrogen excreted. The larvae of the wax moth, Galleria mellonella (L.), excrete hypoxanthine and xanthine, although uric acid is the major nitrogenous metabolite (Nation and Patton 1961, Nation 1963). Quantitative information concerning these three purines in Galleria would clearly be of value in assessing the possible effects on water conservation, and also might be expected to yield fundamental information concerning the physiology of nitrogen metabolism. There are several methods for purine analysis available, but a careful study of their suitability for use on crude preparations of insect tissue is needed. A method should be specific and accurate at the microgram level, for volumes of haemolymph or excretory material available from many insects are exceedingly small. The quantitative methods reported upon in this paper are well known methods which have been used successfully to estimate purines in a variety of biological material. The purpose of the present study has been to determine which, if any, of these methods are suitable to estimate purines in insect tissues and excreta. The colony of Galleria was started from field collections of larvae near Gainesville, Florida, and maintained in the laboratory on Haydak's diet (1936). Hypoxanthine, xanthine, and uric acid were purchased from Nutritional Biochemicals Corporation. Folin phenol reagent was purchased from Curtin and Company.

The Effects of Coenzyme A upon the Oxidation of Epinephrine and Norepinephrine

Coenzyme A was obtained from the Nutritional Biochemicals Corporation. Comparison of the measured and theoretical content of the sulfhydryl group (3) indicated that 44% by weight of this preparation was coenzyme A. The I-bitartrate forms of epinephrine and norepinephrine were obtained from the Winthrop-Stearns Company. Solutions containing 100 pg of a catecho1 amine per ml of 0.01 N HCl were kept in the refrigerator, and aliquots were taken for daily use. The tyrosinase came from the Worthington Biochemical Corporation. Separate solutions containing 2.3

Digestion of collagen and reticulin in paraffin sections by collagenase.

Tissues are fixed in ethanol or in Carnoy's 6:3:1 mixture and embedded in paraffin after routine ethanol dehydration. Sections are taken to water and then covered with 0.2 ml of a 0.9% NaCl solution containing 1 mg/ml of collagenase, and incubated at 50° C for 45 min. After this, they were washed and then stained by the usual methods for connective tissue fibers. Control sections were made by substituting plain 0.9% NaCl solution for the collagenase solution. The collagenase used was from bacteria and obtained from Nutritional Biochemicals Corporation, Cleveland 28, Ohio.

The demonstration of photochemical reducing sites in Chromatium sp

DYAn [I] used blue tetrazolium to demonstrate intracellular particles exhibiting marked reducing activity in illuminated and in non-illuminated plant tissues. The blue-black reduced tetrazolium appeared in granules dispersed throughout the cytoplasm of blue-green algae and within the intracellular chloroplasts of green algae and higher plants. Since the dye was reduced more rapidly and at more numerous sites in illuminated than in non-illuminated cells, she concluded that these stained particles probably represented grana. We have demonstrated similar sites in isolated bean leaf chloroplasts [4]. The present investigation was undertaken to adapt the blue tetrazolium technique to the study of the photosynthetic bacterium, Chromatium sp., and to demonstrate, if possible, the intracellular loci of photochemical reduction. The preliminary results reported here indicate that this technique is more convenient and may prove to be more specific and more reliable than growth or respirometric methods for studying the photoskythetic and dark metabolism of specific substrate by these organisms. Cells were grown in liquid media containing either sodium sulfide or sodium thiosulfate [3], harvested by centrifugation, washed twice with and finally resuspended in 0.01 M phosphate buffer, pH 7.0. The indicator solution was prepared by dissolving 1 mg of tetrazolium blue (Nutritional Biochemicals Corporation) in 30 ml of 0.03 M phosphate buffer, pH 7.0. Solutions of substrate were prepared in tap water and contained either 0.5 per cent Na,S * 9 H,O or 1.0 per cent Na,S,O, * 5 H,O. One ml of the indicator solution was mixed in a Thunberg tube with 1 ml of a IO per cent suspension of cells and 1 ml of either tap water or substrate solution. The tubes were evacuated for three minutes with a water aspirator and sealed. All tubes were prepared in duplicate. One series was placed in a water bath at 27°C and illuminated from below with a loo-watt incandescent bulb in a reflector desk lamp. The second series was completely wrapped with aluminium foil to exclude light and placed in the same bath. The incubation period was varied from I to 24 hours. Although we have not been able to prevent photoreduction of tetrazolium in illuminated cells lacking exogenous subtrate, we have succeeded in delaying for more than an hour any detectable evidence for the reduction by using cells which had been depleted of interfering endogenous materials (starved cells). Usually, intense illumination for two hours was sufficient to starve buffered suspensions of cells which had been harvested from thiosulfate medium. Cells grown in sulfide medium normally contained intracellular sulfur droplets which disappeared only after several days of illumination. Consequently, sulfid-grown cells were not starved for the tetrazolium tests.

Decarboxylation of Pyruvate by Thiamine Analogues

Sources of Compounds Tested-Thiamine hydrochloride (Merck, U.S.P. grade), 0-acetylthiamine hydrochloride and oxythiamine dihydrochloride (California Foundation for Biochemical Research), pyrithiamine hydrobromide and 4-amino-5 aminomethyl-a-methyl pyrimidine dihydrochloride (Nutritional Biochemicals Corporation) were all used without further purification. For determination of pK, values, the 4-amino-5-aminomethyl-2-methyl pyrimidine dihydrochloride was recrystallized from ethanol and ether until it gave the theoretical end point when titrated with base. 5-(2-Hydroxyethyl)I-methyl thiazole was redistilled from pooled commercial products of Merck’ and du Pont. The boiling point was lo%‘, 0.8 mm. Infrared inspection of the redistilled product showed the loss of a strong carbonyl peak which was originally present. The purified product was stored in a desiccator at -20” to hinder oxidation. Salts of 5-(d-Hydroxyethyl)-4-methyl Thiazole-The method of Clarke (6) was employed to prepare the 3-methyl and 3-benzyl (7) and the 3-o-, m-, and p-nitrobenzyl (8) salts. The 3-cr-methylbenzyl salt was prepared by dissolving 0.04 mole each of oc-bromoethyl benzene (Eastman, redistilled 89”, 15 mm.) and 5-(2-hydroxyethyl)-4-methyl thiazole in 4 ml. of thiophene-free anhydrous benzene in a stoppered 18 x 150-mm. test tube. The solution was heated overnight at 55”. The amethylbenzyl salt separates as a light yellow syrup. Attempts to crystallize this compound from a number of solvent systems were all unsuccessful. During crystallization attempts in hot absolute ethanol the salt was partially solvolyzed to the pyrimidylmethyl ethyl ether and 5-(2-hydroxyethyl)-4-methyl thiazole hydrobromide. The absorption spectra in both acid and base and the titration behavior very closely paralleled those of the 3-benzyl salt. These properties plus the method of preparation leave little doubt as to the character of the product. The 3-cyanomethyl salt was prepared in a similar manner from chloroacetonitrile (Eastman white label, redistilled 24O, 27 mm.) and 5-(2-hydroxyethyl)-4-methyl thiazole. The solution was heated at 80’ for 3 days, the benzene supernatant was discarded,

The Purification and Properties of Glutathione Peroxidase of Erythrocytes

phenylhydrazine and hydrogen peroxide were prepared fresh as needed for the various experimental procedures. The hydrogen peroxide concentration was determined by titration with a standardized permanganate solution. Horseradish peroxidase was obtained from Nutritional Biochemicals Corporation. Diethylaminoethyl cellulose1 was prepared from Solka-Floe as described by Peterson and Sober (8). Titration curves of the lyophilized preparation showed the presence of 0.9 m.eq. of ionizing groups per gm. of dry material. Methods-All spectrophotometric measurements were made with a model DU Beckman spectrophotometer. Optical density readings at 270 rnp were used as a routine measure of the protein concentration in eluent fractions. The optical density readings at 270 rnp of a solution of the purified enzyme preparation were correlated with the protein nitrogen content of this same sample by a nitrogen analysis carried out by the Kjeldahl procedure (9). Catalase activity was measured by the method of Feinstein (10).

A Minced Chicken Embryo Medium for Cultivating Pleuropneumonia-Like Organisms

The pleuropneumonia-like organisms (PPLO) of animal origin require tissues or body fluids for growth (Edward, 1954). Minced chicken embryos also provide substances capable of supporting growth of PPLO (Nelson, 1939; Lecce, Stinebring and Morton, 1953).It is the purpose of this note to describe a simplified medium composed of readily available ingredients for the cultivation of PPLO of animal origin. The medium has the following composition: Phenol Red Broth Base (Difco) 1.6 percentYeast Hydrolysate (Nutritional Biochemicals Corporation) 1.0 percentGlucose 0.5 percentMaltose 0.5 percentThallium Acetate 0.025 percentThe above basal medium was autoclaved and adjusted to pH 7.8 with: 0.1 N sodium hydroxide Minced Chicken Embryos (Heated for 3 min. at 100°C.) 1.0 percentPenicillin (Added aseptically) 1000 units/ml.The basal medium was prepared in 100 ml. quantities and autoclaved for not more than 10 min. at 121°C. Minced embryos were obtained by macerating 7-day-old whole washed …

An evaluation of thymidine in treatment of tropical sprue.

SummaryInjections of a potent preparation of Thymidine in doses equivalent to at least 20, 47, and 80 μg of Vit. B12 were without effect in producing hematologic improvement in patients with tropical sprue in relapse.The thymidine used in this study was purchased from Nutritional Biochemicals Corporation.