Correlation between pregnancy diagnosis by membrane slip and embryonic mortality.

In a study involving 7,477 pregnant cows (5,426 Holstein-Friesians and 2,051 Guernseys), each pregnancy was diagnosed by rectal palpation, using the membrane slip technique. Each cow was examined twice, first by a veterinary student, then by a clinician. Of the cows diagnosed pregnant, 7,058 (94.4%) calved. The embryonic death rate in cows examined during the first 50 days of gestation was significantly (P less than 0.001) greater than that associated with examinations after 50 days of gestation. A significant difference was found between herds in different years (P less than 0.05). There was no conclusive indication that embryonic loss at the time of or shortly after early examination was iatrogenic abortion resulting from the membrane slip technique.


Agammaglobulinemia in a horse.

Immunologic deficiency was suspected in an 18-month-old Standardbred horse with persistent fever, multifocal bacterial infection, and neutropenia with a large number of immature neutrophils. Serum protein electrophoresis revealed marked depression of the gamma-globulin fraction (0.2 g/100 ml). Immunologic testing and histologic examination of lymphoid tissues identified the immune deficit as agammaglobulinemia. Serum concentrations of immunoglobulin (Ig)G and IgG(T) were initially low and declined with time; IgM and IgA were not detectable. The horse failed to produce antibodies when inoculated with foreign antigens but had a positive cell-mediated skin reaction to intradermal phytolectin injection, and lymphocytes responded normally to in vitro stimulation by mitogens. Histologic examination of lymphoid tissues revealed absence of germinal centers and plasma cells.


A quantitative computerized recording method in clinical education: a pilot project.

This paper describes an approach to a quantitative computerized method for recording the clinical education experiences of physical therapy students. While the project was conducted in the field of physical therapy, it provides a model that can be adapted to other allied health fields. The objectives, methodology, outcome and future prospects of the project are discussed.


Retrograde axon reaction in the lateral vestibular nucleus of neonatal and adult rats.

Upper cervical cord lesions were performed in neonatal and adult rats (neonates survived for 10-20 days, adults 7-30 days). Retrograde axonal reaction was examined in the lateral vestibular nucleus. Nuclear eccentricity, which is a normal feature in neonatal lateral vestibular neurons, is not peculiar to adult neurons. As early as 7 days after cordotomy, neonatal neurons reacted severely, to the extent of atrophy and cell loss. Seven days after cordotomy, adult neurons were swollen, with eccentrically placed nuclei and Nissl granules which had undergone dissolution. The latter feature is regarded as the diagnostic feature in identifying reacting chromatolytic cells.


A comparison of the processes of ventricular coarctation and choroid and ependymal fusion in the mouse brain.

Lateral ventricular coarctation and fusion of the choroid and ependyma was studied in prenatal and postnatal mice. Coarctation of the lateral ventricle was preceded by the presence of a large quantity of extracellular glycogen embedded in an electron-dense, amorphous matrix lying between the ventricular walls. As the site of coarctation is approached the quantity of glycogen and ground substance decreases and eventually the cilia and microvilli of the ependymal cells also disappear and adjacent walls are joined by maculae adherentes. The glycogen may be produced by the choroid plexus. The presence of cilia seems necessary for coarctation to occur. Fusion of the choroid plexus with the ependyma appears to be due simply to entanglement of choroidal microvilli and ependymal cilia without any specialised cell to cell connexions being present.


Arterial segmentation in the spleen of the sheep (Ovis aries).

Forty corrosion casts of the splenic artery and its tree were used to study segmentation in the sheep spleen. Twenty eight spleens (70%) had two arterial segments, a right and a left. One spleen (2.5%) had a visceral and a parietal segment. Eleven spleens (27.5%) showed no arterial segmentation, the artery ramifying irregularly.


Isolation and characterization of endothelial cells from rat and cow brain white matter.

A procedure is described for obtaining pure cultures of endothelial cells from rat and cow brain white matter. The morphology of the tissue-cultured cells was studied by both light and transmission electron microscopy. Weibel-Palade bodies, described in the literature as specific for endothelial cells, appeared in small numbers in our cultured cells. Autoradiographic, scintillation counting, immunological and histochemical studies were performed. The usefulness of pure endothelial cell cultures is discussed.


Alterations in liver dehydrogenases during tail regeneration in the gekkonid lizard, Hemidactylus flaviviridis.

Histochemical studies on the activities of alpha-GPDH, LDH, SDH and MDH in liver have been carried out during the different phases of tail regeneration in the lizard, Hemidactylus flaviviridis. Changes in the metabolic activities of the liver during regeneration indicate that during the initial phases of regeneration (namely, wound healing and blastema formation) the energetics of the hepatic tissue are anaerobically oriented, but later (i.e. during the growth phase) the TCA cycle appears to be predominant.


Fibre number and fibre size in a surgically overloaded muscle.

Soleus muscles of male and female mice were overloaded by surgical resection of parts of gastrocnemius and plantaris. The effects of overloading were examined histologically after 7, 55 and 208 post-operative days, and also in teased preparations. Animals studied after 7 post-operative days showed a marked increase in muscle weight, but no significant change in mean fibre diameter or fibre number. Animals studied after 55 and 208 post-operative days showed an increase in soleus muscle weight, with fibre hypertrophy (but no increase in fibre number) proximally, while distally there was an increase in the number of fibre profiles in cross sections, some being wider, some thinner than normal. The small diametered fibres seem to persist indefinitely. From the evidence, both direct and indirect, it was concluded that surgically overloaded fibres split longitudinally into unequal parts, and that this explains the increase in fibre profiles in distal cross sections as well as their variation in size. It is clear that, because of the splitting, a surgically overloaded muscle is a difficult model on which to study fibre hypertrophy.


The time scale of tooth development and replacement in Xenopus laevis (Daudin).

One hundred and seventy two larval specimens of Xenopus laevis were reared in such a way that their rates of development (as measured by external criteria) were similar, and so the course of dental development could be examined histologically in a cross sectional study. In this way the events of tooth development were observed, and a time scale constructed for these events. The teeth took an average time of 26 days to develop, erupt and become ankylosed to the bony pedestal, after which each tooth was in a functional position for only about 7 days. Individual tooth replacement was assessed to occur about every 16 days. By comparing the number and size of the odontoblasts responsible for dentinogenesis in 18 months old adult Xenopus laevis with the odontoblasts in the larval specimens, the conclusion was drawn that, despite the larger size of the adult teeth, the time involved in their development and replacement may well be of similar duration to the smaller larval teeth. The significance of the findings for the Zahnreihen Theory is discussed.


Torsion in metacarpal bones and bilateral asymmetry.

A comparison of the relative position of the dorsoventral axes of the bases and heads of the metacarpal bones shows that these do not lie in the same plane, and that torsion can therefore be said to exist in these bones. Torsion is such that the heads of the second and third metacarpals appear to be rotated laterally relative to the base, whereas the heads of the fourth and fifth metacarpals appear to be rotated medially. The usual direction of "rotation" of the head of the first metacarpal is to the lateral side in the right hand and to the medial side in the left hand (P less than 0.001). In addition, there are statistically significant differences in the degree of torsion in other metacarpal bones of the right and left sides: torsion in the second and third metacarpals is greater in the right hand (P less than 0.001), while torsion in the fourth and fifth digits is greater in the left hand (P less than 0.001). All these asymmetries represent a "lateral shift" in the degree of torsion in metacarpal bones of the right side. Torsion in the metacarpal bones appears to facilitate efficiency of the grip: it is apparently an important factor in preventing crowding together of the second to fifth digits during flexion.


Ossification and mineralization in the tendons of the chicken (Gallus domesticus).

Fifty broiler chickens of both sexes were used in this study. The chickens were bought at 8 weeks old and kept until 40 weeks old. During this period the development of ossification and mineralization was followed. The chickens were killed at weekly intervals. After having been examined fresh, the tendons were fixed in appropriate fixatives and processed for histological and histochemical examination. The earliest signs of mineralization were noted at 15 weeks and mineralization was apparent on X-ray photographs at 20 weeks. The development of ossification of the tendons has been described. The fibroblasts transformed into osteoblasts which secreted around the cells and later between the fibres. This was followed by the appearance of osteoblasts and resorption cavities and then the latter became lined by osteoblasts, and osteones were formed.


Embryo surface morphology during post-gastrula development of the frog, Bombina orientalis, as revealed by scanning electron microscopy.

To further study the morphology of the developing amphibian embryo, and to provide a basis for the characterization of abnormal embryogenesis, surface features of the Bombina orientalis embryo during post-gastrula development were examined with the scanning electron microscope. Ciliated cells similar to those reported for Rama pipiens and Ambystoma mexicanum were observed in the epidermis. Epidermal secretory cells displaying large numbers of exocytotis apertures become prominent at the early tail bud stage and are prevalent through hatching. The surface features of the adhesive organs and gill filaments are also described. Removal of the embryo from the fertilization envelope resulted in the accumulation of a prominent mucoid matrix over the surface cells. These findings should be considered when experiments are designed which involve the effects of applied molecules on amphibian embryo epidermal cells.


Morphological observations on the mesonephros in the postnatal opossum, Didelphis virginiana.

The mesonephros of the opossum persists for 3-4 weeks into the postnatal period. Based on our observations of its structure, and the vital dye experiments of others, it appears that the opossum mesonephros is functional during the first 10 days of the postnatal period. The mesonephros of the newborn opossum consists of 35-45 nephron units which are structurally very similar to metanephric nephrons except that they lack a loop of Henle. By the end of the first postnatal week, regression which proceeds in a craniocaudal direction, is observed. By the end of the second week most nephrons show some signs of regression. The regressing mesonephric nephrons are replaced by connective tissue.


Effects of tenotomy on the motor end plates of fast and slow twitch muscles of the rabbit.

The projection areas of vitally stained motor end plates in normal and tenotomized fast and slow twitch muscles of the rabbit hind limb were measured. There was a significant reduction in the mean projection area of the motor end plates of the slow muscle after tenotomy. There was no significant change in the projection area of the FDL or gastrocnemius motor end plates after tenotomy. After tenotomy the deep (slow) component of the tibialis anterior showed an increase in the mean projection area of its end plates and so approached the faster superficial component in this respect. It appears therefore, that the altered pattern of activity of the slow motoneurons (which is known to occur after tenotomy) is associated with a change in the end plate size towards that normally found in muscles innervated by phasic motoneurons.


Efferent connexions of the pars lateralis of the substantia nigra (SNL).

Unilateral lesions were made in the lateral nucleus of the substantia nigra in eight cats and in the zona compacta and reticularis in two cats. After a 10 days survival period, brains were perfused and stored in 10% formalin. Frozen sections were stained with the De Olmos-Ingram and Fink-Heimer silver methods for degenerating axons and terminals. Efferents from the lateral nucleus of the substantia nigra projected to the dorsolateral third of the head of the caudate nucleus, putamen, medial reticular formation, central tegmental tract, amygdala, zona compacta and reticularis of the substantia nigra, ventral anterior and ventral ateral thalamic nuclei. Projections to the amygdala, medial reticular formation and central tegmental tract were seen only in animals with lesions in the lateral nucleus of the substantia nigra. Projections to the caudate and putamen were more abundant when compared to those in the thalamus. Within the thalamus, projections to the ventral lateral nucleus were scanty compared to those in the ventral anterior nucleus. The present study compares for the first time efferent connexions of the lateral nucleus of the substantia nigra and those of the pars compacta and reticularis. The findings suggest a specialization of the different zones of the substantia nigra with regard to their projection sites.


Calvarial and sutural re-development following craniectomy in the neonatal rabbit.

In the present investigation extensive calvariectomy was performed on neonatal rabbits and the subsequent regeneration of the calvarium was followed grossly, radiographically and histologically. The results indicate that regeneration of the calvarium is a gradual process which exhibits a definite pattern and rate. Furthermore, although the sutural articulations re-develop in their proper anatomical positions and exhibit normal histological structure, they tend to synostose earlier than controls. Of perhaps greater biological significance is the apparent importance of the periosteal envelope during the regenerative process. Maintenance of dural integrity during operation, and the re-establishment of pericranial continuity post-operatively, seem to be essential for restoration of normal sutural and skeletal architecture.


Cell death during the development of the truncus and conus of the chick embryo heart.

The presence of cell death in the walls of the truncus and conus of the developing chick heart was investigated by a variety of light and electron microscopic techniques. Necrotic areas were observed in the myocardial layer of the truncus and conus and within the mesenchymal cells of the truncoconal ridges and aortopulmonary septum. These necrotic zones appeared first at Stage 25-26 and reached their maximum extent at Stages 29-32 undergoing later progressive disappearance. The morphological changes of the degenerating cells detectable under both transmission and scanning electron microscopy are also reported. The possible role of cell death in the morphogenesis of the truncus and conus is discussed.


Neoxaline, a new alkaloid produced by Aspergillus japonicus. Production, isolation and properties.

A new alkaloid named neoxaline has been isolated from culture broth of Aspergillus japonicug Fg-551 by solvent extraction and silica gel chromatography. The compound does not possess antimicrobial activities, but weakly stimulates the central nervous system. The molecular formula of neoxaline has been determined as C23H25N5O4 on the basis of elemental analysis and its mass spectrometry.


Herquline, a new alkaloid produced by Penicillium herquei. Fermentation, isolation and properties.

A new alkaloid named herquline has been isolated from culture broth of Penicillium herquei Fg-372 by solvent extraction and silica gel chromatography. The molecular formula of herquline has been determined as C19H26N2O2 on the basis of elemental analysis and its high resolution mass spectrometry. The compound does not possess antimicrobial activities, but weakly inhibits blood platelet aggregation induced by adenosine diphosphate.


Antitumor anthracycline antibiotics, aclacinomycin A and analogues. I. Taxonomy, production, isolation and physicochemical properties.

Aclacinomycin A and B, two major components of a new antitumor antibiotic complex, and their 19 analogues were produced by a culture of strain No. MA144-M1, which was identified as Streptomyces galilaeus. They were isolated by chelation with copper ion and silicic acid chromatography, and characterized by physicochemical methods in the anthracycline group of antibiotics.


Preparation of delta 3-7 alpha-phenylacetamidodesacetoxycephalosporanic acid.

delta 3-7 alpha-Phenylacetamidodesacetoxycephalosporanic acid was prepared by ring expansion of 6-epi-benzylpenicillin-S-sulfoxide, using N,O-bis(trimethylsilyl)acetamide (BSA) as silylating and dehydrating agent and alpha-picoline/alpha-picoline hydrobromide as catalyst. In some experiments 7 alpha-phenylacetamido-3 beta-bromo-3 alpha-methylcepham-4 alpha-carboxylic acid was obtained as a side product. 7-Epimers in the desacetoxycephalosporanic series were also prepared by base-catalyzed epimerization of the benzyl 7 beta-(p-nitrobenzylideneimino)desacetoxycephalosporanate and of the S-sulfoxide of natural methyl 6-phenylacetamidodesacetoxycephalosporanate. In both reactions 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) was used as epimerization catalyst.


DNA strand scission induced by adriamycin and aclacinomycin A.

The binding of adriamycin and aclacinomycin A with PM2 DNA, and the consequent cleavage of DNA have been demonstrated by agarose gel electrophoresis, using an ethidium bromide assay. Adriamycin was observed to induce a single strand scission of DNA in the presence of a reducing agent, but aclacinomycin A caused much less degree of DNA breaks. The DNA cleavage was enhanced by Cu2+ and Fe2+, but not significantly by Ni2+, Zn2+, Mg2+ and Ca2+, suggesting that reduction and auto-oxidation of the quinone moiety and H2O2 production participate in the DNA-cutting effect. The DNA degradation was dependent upon concentrations of the anthracyclines and CuCl2. The degree of DNA cleavage at 0.04 mM adriamycin was similar to that at 0.4 mM aclacinomycin A in the presence of 1 mM NADPH and 0.4 mM CuCl2. DNA was degraded to small fragments at 0.4 mM adriamycin and 0.2 mM CuCl2. The anthracycline-induced DNA cleavage was stimulated by H2O2, but partially inhibited by potassium iodide, superoxide dismutase, catalase and nitrogen gas atmosphere. The results suggested that both free radical of anthracycline quinones and hydroxyl radical directly react with DNA strands.


Amikacin dosage in the preterm newborn.

Twenty-two preterm infants of gestational age 26 to 34 weeks were treated with amikacin for suspected bacterial infection, using a dose of 7.5 mg/kg, 12-hourly, intra muscularly. Blood levels were measured using a radio-immunoassay kit capable of giving results within 4 h. One-hour peak levels showed wide variation and the mean level one hour after the first dose was 18.2 mg/1; in severe infections an initial loading dose of 10 mg/kg is therefore recommended. Trough levels in individual infants also varied considerably from day to day, but showed no overall accumulation. There was no obvious adverse effect on hepatic or renal function.


Prospective comparative evaluation of gentamicin or gentamicin plus cephalothin in the production of nephrotoxicity in man.

Recent studies in animal models have demonstrated that in contrast with humans, cephalothin (CTIN) does not increase gentamicin (GENT) nephrotoxicity, but rather protects against it, particularly when CTIN is given simultaneously with GENT. To investigate this phenomenon in humans a study was designed in which 67 patients suffering from mild infections were investigated. Thirty-three of them served as the control group receiving GENT alone at a dose of 1.5 mg/kg/8 hourly, while the remaining 34 received CTIN at a dose of 2 g or 3 g 8 hourly by i.v. bolus, either simultaneously with GENT or separated by a 4-h interval. Findings showed that: (a) cylindruria developed in 66.6% and 82.3% and 82.3% in the GENT and GENT + CTIN groups respectively, (b) urinary beta-glycuronidase activity increased in 57.5% and 75% (c) serum creatinine exceeded by 0.3 mg the initial values in 21.2% and 27.6% and (d) the blood urea was above 50 mg% in 18.1% and 17.6% of the patients. These results indicate that: (a) regardless of the route and order of administration simultaneous treatment did not protect against nephrotoxicity in humans; (b) the combination of GENTA plus CTIN has no synergistic effect on the production of elevated serum creatinine and rising blood urea; (c) urinary beta-glycuronidase is not a significant predictor of eventual nephrotoxicity; (d) the following risk factors influenced the appearance of nephrotoxicity in both groups: (1) elevated GENT trough levels greater than or equal to 2 mg/l; (2) a course of treatment longer than 10 days.


Effect of food on absorption of erythromycin. A study of two derivatives, the stearate and the base.

Two derivatives of erythromycin, the stearate and the enteric-coated base have been given in a cross-over study to both fasting and non-fasting volunteers. The stearate gave higher plasma concentrations in non-fasting than in fasting subjects. The area under the curve was significantly larger during steady state day 7 than after a single dose. The base showed larger individual variations in concentrations. The release of the drug from the enteric-coated tablets was depressed by food, but the absorption was not prevented.


Spectrophotometric determination of caffeine in coffee products: collaborative study.

An ultraviolet (UV) spectrophotometric method for determining caffeine in regular and decaffeinated coffee products has been studied collaboratively. Nine laboratories participated in this study which compares the proposed UV method with the official AOAC micro Bailey-Andrew method. Caffeine content was determined on as-is basis on 8 samples of green, roasted, and soluble coffees. The coefficients of variation for the proposed method ranged from 2.02 to 6.98% for the 8 samples studied. The results agreed well with those from the Bailey-Andrew Method. The method was adopted as official first action.


IUPAC gas chromatographic method for determination of fatty acid composition: collaborative study.

An international collaborative study of IUPAC methods II.D.19 and II.D.25 for preparation and GLC analysis of fatty acid methyl esters was begun in 1976. The IUPAC methodology, applicable to animal and vegetable oils and fats and fatty acids from all sources, contains special instructions for preparation and analysis of methyl esters of fatty acids containing 4 or more carbon atoms (analysis of milk fat). Twenty-three collaborators participated in the analysis of 5 known mixtures, 4 vegetable oils, 1 fish oil, and 2 butterfats. Several blind duplicate samples were included. The experimental data were subjected to statistical analysis to examine intra- and interlaboratory variation. Reproducibility and accuracy data for the higher fatty acid (14:0-22:1) mixtures and fish and vegetable oils were satisfactory and were in good agreement with results from an AOCS Smalley Committee check sample program involving analysis of the same samples. Typical coefficients of variation (%) at various concentrations were 15 (2% level), 8.5 (5% level), 7 (10% level), and 3 (50% level). Low recoveries and poor reproducibility were characteristics of results obtained for butyric acid in the butterfat and related known mixtures. A coefficient of variation of about 19% was found for analysis of butyric acid in butterfat, vs coefficients of variation in the range of 4-13% for similar levels of other components in butterfat and other samples. The IUPAC methodology for GLC analysis of fats and oils other than milk fats has been adopted by the AOAC as official first action to replace the current GLC method, 28.063-28.067.


Confirmation of organothiophosphorus insecticide residues in fruit and vegetables by oxidative derivatization.

Oxidative derivatization of 10 organothiophosphorus insecticides at nanogram levels, with neutralized sodium hypochlorite (NaOCl) produced their respective P=O oxygen analogs, as shown by GC/MS and FT-IR analysis. In addition, phorate resulted in phorate oxon sulfoxide while methidathion gave a mixture of oxidation products. Four macro-scale oxidation products of methidathion were isolated by thin layer chromatography and 3 were identified, namely, methidaoxon, bis(2-methoxy-delta2-1,3,4-thiadiazolin-5-on-4-yl) sulfide, and the corresponding disulfide, by comparison of their infrared and mass spectra with those of authentic samples. The relative response to sulfide and disulfide products of the flame photometric detector in both the P- and S-modes is discussed. This derivatization is applied to the confirmation of chlorpyrifos, ethion, phorate, DMPA (Zytron), leptophos, and methidathion in celery, potatoes, lettuce, tomatoes, and apples at 0.25-0.5 ppm fortification levels.


Investigation and simplification of the sweep co-distillation cleanup of pesticide residues in animal fats.

The sweep co-distillation technique of Storherr et al. was investigated and simplified. Six organochlorine pesticides in animal fats were cleaned up under various distillation conditions as follows: No solvent was used, or solvent injection rates were 1 mL/min or 2 mL/3 min. Distillation tubes of 6.7 and 9.0 mm id diameter were compared. Distillation temperatures were varied from 150 to 300 degrees C. The complex condenser of Storherr et al. was compared with a simpler pipet condenser. A U-tube condenser which allows direct introduction onto a Florisil column for secondary cleanup was evaluated. The following modifications to the sweep co-distillation technique resulted: no solvent introduction, distillation temperature 230 degrees C, nitrogen flow rate 600 mL/min, 6.7 mm id distillation tubes with simplified packing, and incorporation of the U-tube condenser. The new technique gave recoveries comparable to those of Storherr et al., but it is faster than most bisolvent partition methods and no large volumes of solvent are required.


Flameless atomic absorption spectrophotometric determination of Vendex, an organic tin miticide, in apples, oranges, and tea leaves.

A method is described for the determination of an organic tin insecticide, Vendex or hexakis(2-methyl-2-phenylpropyl)distannoxane (HMPD). Tin compounds are extracted from the sample homogenate with ethyl ether containing 1% acetic acid. HMPD and its degradation products are separated through a silica gel column, and analyzed by an atomic absorption spectrophotometer coupled with a heated graphite atomizer. By this technique, as little as 5 ng HMPD and its degradation products can be determined with an average recovery of 67-96%.


Gas-liquid chromatographic determination of thiabendazole and methyl 2-benzimidazole carbamate in fruits and crops.

A method is described for the electron capture gas-liquid chromatographic determination of thiabendazole and methyl 2-benzimidazole carbamate (MBC) after derivatization with pentafluorobenzyl bromide (PFB-Br). The samples are extracted with ethyl acetate, and the residual thiabendazole, benomyl, and MBC are isolated by liquid-liquid extraction into dilute HCl. After neutralization and re-extraction with ethyl acetate, thiabendazole and MBC are reacted with PFB-Br to form the PFB derivatives. Alumina column chromatography was used to clean up extracts, and the derivatives could be detected as low as 5-10 pg. Recoveries were 95-98% from fruits fortified with 0.3-2.0 ppm thiabenzadole; recoveries were 91-97% when 0.05-1.0 ppm benomyl/MBC were added to fruits/crops. The PFB derivatives were identified by gas-liquid chromatography-mass spectrometry.


Quantitative conformation of dimethoate residues in wheat plants by single ion mass spectrometry.

A gas chromatographic-single ion mass spectrometric method was developed for determining dimethoate residues in wheat plants. The base peak (m/e 37) of dimethoate was chosen as the single ion peak, and methyl stearate was used as an internal standard for this analysis. The minimum detectable concentration of dimethoate by this method was about 0.1 ppm for a 20 g wheat plant sample. The recoveries of dimethoate were about 89% at 0.13 ppm and greater than 96% at 0.5-1 ppm.


Mass spectrometric identification and gas-liquid chromatographic determination of 2-chloroethyl esters of fatty acids in spices and foods.

The 2-chloroethyl esters of 5 fatty acids have been identified in spice and food samples by gas-liquid chromatography-mass spectrometry (GLC/MS). Twenty-four spice samples were analyzed for the 2-chloroethyl esters of fatty acids by AOAC official multiple residues pesticide procedure using GLC with microcoulometric detection. The esters of capric, lauric, myristic, palmitic, and linoleic acids have been identified at levels up to 1400 ppm. 2-Chloroethyl linoleate was the most abundant ester in all samples. Several foods analyzed by the same procedures showed levels of 2-chloroethyl linoleate as high as 35 ppm. Recoveries from fortified samples ranged from 84 to 98% for the various esters. A method using an acid-catalyzed esterification reaction was developed to rapidly determine the fatty acid content of these spices. GLC analysis with microcoulometric detection was used. Recoveries from fortified samples ranged from 92 to 110%. After 2 spice samples found to be free of 2-chloroethyl esters were fumigated with ethylene oxide, the level of 2-chloroethyl linoleate reached 77 ppm. All levels of 2-chloroethyl esters were confirmed by GLC/MS.


Identification of some benzodiazepines of forensic interest.

Ten benzodiazepines--clorazepate, nitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, N-desmethyldiazepam, diazepam, prazepam, and flurazepam--in solid dosage form are identified by ultraviolet (UV) and infrared (IR) spectrophotometry and by high pressure liquid chromatography (HPLC). UV absorption data and IR spectra of the 10 benzodiazepines are provided and HPLC separation methods are described.


Identification tests for propantheline bromide.

A method is described for the isolation and identification of propantheline bromide from bulk drug substances and dosage forms, both alone and in combination with other drug substances. The method permits the specific identification of the intact drug substance, using both infrared spectroscopy and thin layer chromatography.


Liquid chromatographic determination of desoxycorticosterone acetate in oil injections.

To determine desoxycorticosterone acetate in oil injections, reverse phase partition chromatography on silanized, purified siliceous earth was used to separate the corticosteroid ester from the bulk of the oil vehicle. The latter was retained on the column while the steroid and the sterol and triterpenoid fractions of the oil were eluted. An internal standard was added to this eluate, which was then subjected to reverse phase high performance liquid chromatography (HPLC). The desoxycorticosterone acetate was quantitatively separated in the HPLC procedure from any free desoxycorticosterone, preservatives, and minor components of the oil. The suitability of the HPLC procedure was verified with a number of C18 packing materials, both pellicular and microparticular. The desoxycorticosterone acetate was adequately resolved from the internal standard, progesterone, with most C18 packing materials evaluated. The proposed procedure provides a suitable stability-indicating assay for desoxycorticosterone acetate in oil injections.


Reverse phase high pressure liquid chromatographic determination of some iodoamino acid contaminants in sodium liothyronine or sodium levothyroxine.

Some iodoamino acids have been separated and determined by high pressure liquid chromatography on octadecylsilane reverse phase packing with a mobile phase consisting of methanol, potassium phosphate monobasic, and orthophosphoric acid at 44 degrees C. The method separates and quantitates mixtures of 3,5-diiodothyronine, liothyronine, isoliothyronine, and levothyroxine. The procedure provides an accurate, sensitive, and rapid estimation of decomposition and/or impurities in standards in approximately 30 min, at the less than 75 pmole level, using an ultraviolet detector at 254 nm.


Teratology.

Two categories of collaborative studies are defined. Examples of each type are presented through teratologic studies conducted at the National Institute for Environmental Health Sciences (NIEHS) in collaboration with an Environmental Protection Agency laboratory and a laboratory in the USSR. These studies and the few pertinent studies found in the literature point out the problem of obtaining repeatability and uniformity of results among tests conducted to determine teratogenic potential. Collaborating laboratories should take more time to select the procedures to be used, to standardize the intended protocol in greater detail, to train participating personnel uniformly, and to test for repeatability by use of a known teratogen, before tackling major interests. Establishment of an archives would improve duplication of published results by making available additional pertinent unpublished information. Finally, possible advantages of collaborative studies for the testing of teratogenic potential are offered.


Interlaboratory comparison of behavioral testing.

New requirements by several regulatory agencies for testing the psychotoxic potential of new drugs, chemicals, and environmental contaminants raise unique problems. In order to assess intra- and interlaboratory reliability of behavioral tests a model animal maze learning procedure was designed and run in 3 cooperating laboratories. Uniform procedures were written and identical mazes were constructed. Normal control animals of identical age and sex, but of different strains, were used by the participants. A positive control group of neurologically impaired rats was run by one laboratory. Significant differences in test results among the laboratories were found. Data obtained from the positive control animals (mean errors=28.3) indicated a learning impairment statistically significant compared to the negative control data (mean errors=12.7) from any of the participating laboratories. Based on the results of this study, a reasonable standard of interlaboratory reliability in behavioral testing appears an attainable goal.


Collaborative studies on cytogenetic and dominant lethal assays.

Two in vivo cytogenetic studies and one dominant lethal study were jointly conducted by members of government, academic, industrial, and independent commercial laboratories between 1970 and 1975 to determine the validity and reproducibility of the techniques. The cytogenetic studies consisted of preparations and analyses of bone marrow cells from male rats for chromosome abnormalities. In the first study, participants from 4 laboratories jointly prepared slides at a Food and Drug Administration (FDA) laboratory according to a prescribed procedure and independently analyzed the slides in their own laboratories. In the second study involving 6 laboratories, a workshop was held at Dow Chemical Co. to reduce scoring differences and to develop a joint protocol. All participants then independently performed the exact procedure in their own laboratories, using animals from a common source. In the dominant lethal study involving 6 laboratories, a workshop was held at the FDA to discuss protocol and to practice the technique. All participants then independently conducted the study in their own laboratories using rats and test chemicals from common sources. In all 3 studies, significant interlaboratory variability existed, and the differences varied with the parameters analyzed.


Intercomparison study on the determination of single administration toxicity in rats.

In 1977, the Commission of the European Communities initiated an intercomparison study to determine the single administration oral LD50 value in rats of each of 5 chemicals with the aims of comparing experimentation technologies, determining the degree of variation in the results and the various parameters used to establish the LD50 value, and establishing a common protocol for the determination of the LD50 value. Sixty-five laboratories in 8 countries took part in the first study. The significant variation in protocol may have led to the large interlaboratory variation observed in the results. Therefore, participating laboratories carried out a second study using a common protocol, preceded by a pilot study. A total of 100 laboratories in 13 countries participated. A majority of results of the second study are currently being analyzed, and the indications are that the interlaboratory variation has been significantly reduced.


Comparison of studies on saccharin and sodium nitrite.

A review of long term animal studies of saccharin and sodium nitrite was undertaken to assess the effect of variability of selected protocol elements on the results obtained. These elements were divided into 4 general categories: design, including selection of test animals, basal diet, dosage form and doses of test substance, route of administration, and duration of exposure; observations, including gross observations during life and at necropsy, clinical tests, and histopathology; performance, including conduct of the test and animal husbandry; and analytical procedures, including chemical and statistical analyses. Because many of the protocol elements are not fully discussed in study reports, it was often impossible to determine what actually had been done. The review of various saccharin studies suggests that bladder tumors resulted following in utero exposure. In utero exposure with sodium nitrite did not appear to cause reticuloendothelial changes. The numerous variations in protocol elements in the nitrite studies precluded identification of a prime element responsible for the variation in reticuloendothelial changes observed. It can be concluded from this review that achievement of reproducibility in long term studies requires minimal variation of protocol elements for the new study.


Comparison of carcinogenicity studies with aldrin and dieldrin.

Laboratory testing of animals is the principal method used to identify chemical carcinogens. However conflicting results are often obtained. This is emphasized in a cross-comparison of 6 aldrin-dieldrin studies; 3 were positive for tumor induction and 3 were negative. Four components of the assays are examined: husbandry, compound administration, observation, and interpretation of results.


Sensitive thin layer chromatographic detection of high fructose corn sirup and other adulterants in honey.

A highly sensitive procedure has been developed to detect the undeclared addition of high fructose corn sirup (HFCS) to honey. Carbohydrates must be separated first to achieve the requisite degree of sensitivity; charcoal-Celite chromatography was used to isolate a fraction containing oligo- and polysaccharides. The fraction was then concentrated and examined by thin layer chromatography on silica gel. Pure honeys yielded only 1 or 2 blue-grey or blue-brown spots at Rf values greater than 0.35; a series of spots or blue streaks extending from the origin characterized adulterated samples. The method detects HFCS and conventional honey adulterants at levels as low as 10% or less of the total mixture. In addition, the procedure detects the presence in honey of all starch-derived sugar sirups tested thus far, regardless of the plant source.


Charcoal column/thin layer chromatographic method for high fructose corn sirup and spectrophotometric method for hydroxymethylfurfural in honey: collaborative studies.

A new spectrophotometric method is described for determining hydroxymethylfurfural in honey in which interfering background absorption of honey is corrected for by use of a bisulfite-treated sample as blank. Two procedures for detecting high-fructose corn sirup (HFCS) in honey were also tested. In one, charcoal column pretreatment is used to concentrate trace oligosaccharides, followed by thin layer chromatography to differentiate those of HFCS from those of honey. The other method depends on measurement of the isomaltose/maltose ratio by gas-liquid chromatography. The charcoal/thin layer chromatographic method for HFCS has been adopted official first action. The bisulfite method for hydroxymethylfurfural has been adopted interim first action.


Gas-liquid chromatographic determination of adipic acid in crackling candy and soft drinks.

A procedure was developed for the simple and rapid determination of adipic acid in crackling candy and also in soft drinks. An alkaline solution of sample was extracted with ethyl ether to remove fatty substances, and H2SO4 was added to water layer to adjust the pH to less than 2. The acidified layer was saturated with NaCl and then extracted with ether. After drying, the ether layer was concentrated and the adipic acid in the concentrate was methylated using the diazomethane methograph equipped with a flame ionization detector. Recovery of adipic acid from crackling candy and from 2 kinds of soft drinks that had been fortified at the 200 ppm level was 96%. An interlaboratory test was carried out on the determination of adipic acid in orange soft drink. The results obtained by 6 laboratories were between 91 and 100% compared with the theoretical value.


Gas-liquid chromatographic determination of sorbic acid and sodium benzoate in table sirup.

A gas-liquid chromatographic method is described for the simultaneous determination of sorbic acid and sodium benzoate in table sirup. The preservatives are extracted from acidified sirup with ethyl acetate and are analyzed on a gas chromatograph equipped with a flame ionization detector and a glass column (4 ft X 4 mm id) containing 9% SP-1200 and 2% H3PO4 on Chromosorb W (AW). Coefficients of variation for sorbic acid and sodium benzoate are 0.62 and 0.41%, respectively. Analysis time is less than 20 min, with recoveries exceeding 90%.


Aflatoxigenic potential of dried figs, apricots, pineapples, and raisins.

Spores from 3 aflatoxigenic strains were applied to the surface of dried figs, pineapple slices, apricots, and raisins. Half of the samples of each foodstuff was autoclaved (cooked) before inoculation. All 3 strains produced some toxin but not in all of the foodstuffs. No aflatoxins were found in any of the raisin cultures. The overall potential for toxin production in the dried fruit was apricot greater than fig greater than pineapple greater than raisin. Among the cooked substrates, the order was apricot greater than pineapple greater than fig=raisin. For the raw dried fruits, the ranking was fig greater than pineapple greater than apricot greater than or equal to raisin. Pineapple was considered the best broad spectrum substrate of the foodstuffs evaluated in the study.


Products of endogenous N-acetylglucosaminyltransferase activity of Dictyostelium discoideum during growth and early development.

In the presence of Mn2+ and uridine diphosphate-N-acetyl-D-[14C]glucosamine, a total particulate fraction prepared from Dictyostelium discoideum amoebae catalyzed the transfer of N-acetyl[14C]glucosamine to endogenous membrane protein acceptors. No transfer to lipid acceptors was evident. The 14C products obtained from growth-phase and aggregation-competent amoebae were converted to glycopeptides by pronase digestion. The respective glycopeptides appeared identical in their chemical and chromatographic properties, suggesting that the same activity was functioning in both growing and differentiating cells. The results provided no evidence for developmental regulation of this activity in D. discoideum.


Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.

Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase.


Menaquinone (vitamin K2) biosynthesis: conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid by Mycobacterium phlei enzymes.

The coenzyme A (CoA) and adenosine 5'-triphosphate-dependent conversion of o-succinylbenzoic acid (4-[2'-carboxyphenyl]-4-oxobutyric acid) to 1,4-dihydroxy-2-naphthoic acids is an important step in menaquinone (vitamin K2) biosynthesis. Cell-free extracts catalyzing this conversion, obtained from Mycobacterium phlei, were separated into three protein fractions by treatment with protamine sulfate. The second fraction (fraction B) and the supernatant (fraction S) alone did not catalyze dihydroxynaphthoate formation, but did so in combination. All of the results were consistent with the formation of an unstable intermediate, likely an o-succinylbenzoyl-CoA compound, by the action of fraction S. Adenosine 5'-triphosphate was required in this reaction and adenosine 5'-monophosphate was formed. This enzyme activity was termed o-succinylbenzoyl-CoA synthetase: the enzyme showed a marked stability to 0.1 N hydrochloric acid. The presumed o-succinylbenzoyl-CoA derivate was rather unstable; under a variety of conditions, it was converted to a spirodilactone form of o-succinylbenzoate. Fraction B contained an enzyme, termed naphthoate synthase, which converted the o-succinylbenzoyl-CoA derivative to 1,4-dihydroxy-2-naphthoate.


Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

Streptomyces coelicolor A3(2) possesses two plasmids (SCP1 and SCP2) that act as sex factors. The plasmid deoxyribonucleic acid isolated from S. coelicolor A3(2) SCP1- strains A617 and A585 had the same molecular weight and endonuclease cleavage pattern as the SCP2 plasmid. The plasmidless strain S18 SCP2- was isolated from the A617 X A585 cross. SCP2 plasmid-containing strains acted as donors of chromosomal markers, whereas the plasmidless strain acted as recipient. The transfer of SCP2+ donor strain markers into the SCP2- recipient occurred at high frequencies (approximately 75%), was unidirectional, was initiated from a fixed region of the chromosome, and had the SCP2 fertility factor transferred first. The introduction of the SCP2 plasmid into a recipient strain greatly reduced the recombination frequency. These fertility properties differed from those previously reported, thereby suggesting that the SCP2 plasmid examined in this investigation may be an additional variant to those described in the literature. The SCP2 plasmid also regulated production of three antibacterial substances and conveyed resistance for S. coelicolor A3(2) strains against growth inhibition by one of them.


Mechanism of acetate synthesis from CO2 by Clostridium acidiurici.

Total synthesis of acetate from CO2 by Clostridium acidiurici during fermentations of hypoxanthine has been shown to involve synthesis of glycine from methylenetetrahydrofolate, CO2, and NH3. The glycine is converted to serine by the addition of methylenetetrahydrofolate, and the resulting serine is converted to pyruvate, which is decarboxylated to form acetate. Since CO2 is converted to methylenetetrahydrofolate, both carbons of the acetate are derived from CO2. The evidence supporting this pathway is based on (i) the demonstration that glycine decarboxylase is present in C. acidiurici, (ii) the fact that glycine is synthesized by crude extracts at a rate which is rapid enough to account for the in vivo synthesis of acetate from CO2, (iii) the fact that methylenetetrahydrofolate is an intermediate in the formation of both carbons of acetate from CO2, and (iv) the fact that the alpha carbon of glycine is the source of the carboxyl group of acetate. Evidence is presented that this synthesis of acetate does not involve carboxylation of a methyl corrinoid enzyme such as occurs in Clostridium thermoaceticum and Clostridium formicoaceticum. Thus, there are two different mechanisms for the total synthesis of acetate from CO2 by clostridia.


Protein turnover in the extreme thermophile Thermus aquaticus.

Protein turnover in the extreme bacterial thermophile Thermus aquaticus was examined in exponential cultures at 75 degrees C. The relative amount of [3H]leucine incorporated into trichloroacetic acid-insoluble material was stable in pulse-chase experiments assayed over 2.5 h. The trichloroacetic acid-insoluble radioactive leucine was stable upon the addition of chloramphenicol, which blocks protein synthesis in T. aquaticus. The specific activity of a phosphate-repressible alkaline phosphatase, investigated in the presence of chloramphenicol, did not decrease. The addition of excess orthophosphate to cultures derepressed for the alkaline phosphatase did not show a marked effect on the specific activity over a 2-h period. On the basis of these four experiments, it does not appear that a high protein turnover rate is essential for the thermophily of T. aquaticus at 75 degrees C.


Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.


Aromatic aminotransferases in coryneform bacteria.

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) are capable of transaminating all three of the aromatic pathway intermediates; prephenate, phenylpyruvate, and 4-hydroxy-phenylpyruvate. Two molecular species of aromatic aminotransferase (denoted aminotransferase I and aminotransferase II) were partially purified from C. glutamicum and B. flavum, whereas a single aromatic aminotransferase was isolated from B. ammoniagenes. In both C. glutamicum and B. flavum, aromatic aminotransferase I and aromatic aminotransferase II have molecular weights of about 155,000 and 260,000 respectively. The two aromatic aminotransferases from C. glutamicum and B. flavum, although exhibiting a similar spectrum of overlapping specificities, differ substantially in substrate preference. Pyridoxal-5'-phosphate is tightly associated with these aminotransferases, since little loss of activity was detected when partially purified enzyme preparations were assayed in the absence of exogenous pyridoxal-5'-phosphate. The aminotransferases are quite sensitive to inhibition by phenylhydrazine. This has practical application when assay of prephenate dehydratase is desired in the presence of aromatic aminotransferase activity since potentially trivial interference can be negated by selective phenylhydrazine inhibition of aromatic aminotransferase activity. At 0.1 mM concentrations of phenylhydrazine, 90% inhibitions of aminotransferase activities were achieved in partially purified preparations of B. flavum and C. glutamicum.


Caulobacter cresentus mutant defective in membrane phospholipid synthesis.

To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.


Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds.

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.


Novel microbial and chemical components of a specific black-band region in the cockroach hindgut.

An area of the hindgut of the cockroach, Eublaberus posticus, is characterized by its black color. This area is the site of accumulation of metal sulfides in the lumen next to the gut wall. In addition to the normal hindgut flora, two unusual procaryotic organisms are seen by both scanning and transmission electron microscopy only in this area of the hindgut. They are (i) a large rod (1.2 by 6 micrometers) with a tuft of polar flagella, many inclusion bodies, and a distinctive complex wall and (ii) an apparently flexible rod with a helically ridged wall. In addition, phagelike particles are described which are apparently infecting gram-positive bacteria attached to the gut wall in the black band area.


Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.


Partition of alkane by an extracellular vesicle derived from hexadecane-grown Acinetobacter.

The enhanced solubility of hexadecane in the growth medium of hexadecane-grown Acinetobacter species has been related to the accumulation of an extracellular vesicular component. The partition of hexadecane was determined by measuring the amount of [3H]hexadecane bound to the vesicular particle. The vesicle was characterized as a phospholipid-rich, lipopolysaccharide-rich particle with a polypeptide composition similar to the outer membrane of Acinetobacter. The accumulation of an extracellular vesicular component that binds hexadecane in the form of a microemulsion represents another example of molecules produced by microorganisms in response to paraffinic substrates.


Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium.

A bipolarly flagellated magnetotactic spirillum containing intracellular chains of single domain-sized magnetite crystals was isolated by applying a magnetic field to sediments from a freshwater swamp. The organism was cultured in a chemically defined medium containing ferric quinate and succinate as sources of iron and carbon, respectively. Nonmagnetic variants of this isolate were maintained in chemically defined medium lacking ferric quinate. In contrast to magnetic cells, these had less iron and lacked measurable magnetic remanence and the intracytoplasmic crystals. In other respects, including moles percent guanine plus cytosine content, growth characteristics, nutrition, and physiology, the two types were similar. The isolate reduced nitrate without accumulating nitrite and produced ammonia during growth. Nitrate or ammonium ions served as a nitrogen source. The organism was microaerophilic and did not grow anaerobically with nitrate in the medium. In chemically defined medium, cells synthesized magnetite only if the initial O2 concentration in the atmosphere of sealed cultures was 6% (vol/vol) or less.


Amphilphilic nature of spiralin, the major protein of the Spiroplasma citri cell membrane.

Spiralin could not be solubilized in the absence of detergents, and it was shown by charge-shift crossed immunoelectrophoresis that this protein was capable of binding detergents under nondenaturing conditions. These properties indicate the amphiphilic nature of spiralin, which therefore should be regarded as an intrinsic membrane protein. The efficiency of mild (ionic and neutral) detergents to solubilize spiralin was as follows: deoxycholate greater than lauroyl sarcosinate, cholate, taurocholate, taurodeoxycholate greater than Triton X-100 greater than Brij 58 greater than Tween 20, indicating that mild ionic detergents were more effective than neutral ones. Solubilization of spiralin was quantitative with sodium deoxycholate. It was also shown that although a membrane protein is not extractable by a given detergent from the membrane, this does not necessarily mean that the protein is not soluble in this detergent.


Fluphenazine outpatient clinics: a pharmacist's role.

Twenty-five schizophrenic outpatients were treated in a pharmacist-administered fluphenazine decanoate clinic for periods of up to 1 year. These patients had a significantly lower rehospitalization rate, a decrease in medication related side effects, and showed improvement in functional capacity. The results suggest that a pharmacist can be clinically beneficial to schizophrenic outpatients and may help to reduce the cost of treatment programs.


Bipolar affective disorder in aged patients.

Bipolar affective illness may be a relatively common missed diagnosis in the elderly. Inaccurate historical data, atypical course of illness and atypical clinical presentation are all sources of diagnostic error. Lithium and tricyclic antidepressants are effective agents in this age group but require close monitoring, with particular attention to their interaction with illness and other medications. A high index of suspicion for bipolar illness is suggested when elderly patients present with depression.


Who is responsible for the delinquent adolescent? A psychiatric and Talmudic perspective.

The concept of the delinquent adolescent is reviewed in terms of definition, culpability, and rehabilitation in concert with psychiatric and Talmudic perspectives. Seven principles under Talmudic Law are discussed which were used by the Court to help determine whether or not an individual was in fact an incorrigible delinquent. The Talmudic concept has evolved that the delinquent child is a product of a disturbed family and a pathological environment. Aberrant behavior was always interpreted in a holistic fashion in the nomenclature of family dynamics. Talmudic tradition emphasizes the necessity to acquire means for reestablishing the intactness of a healthy family. When it is determined that this is not possible in the nuclear family, mandates are given society for placement elsewhere under the aegis of the Court.


Nylidrin HCL in the treatment of symptoms of the aged: a double-blind placebo controlled study.

This study was designed to assess the effectiveness and the safety of nylidrin HCL in the geriatric patient and mild to moderate symptoms of cognitive, emotional and physical impairment. Following a 3 week placebo washout, 60 patients received either nylidrin HCL or placebo for 12 weeks. Efficacy evaluations were made utilizing the Sandoz Clinical Assessment Geriatric (SCAG) Scale, a nurse's rating of ward behavior (SCASNO), the Hamilton Psychiatric Rating Scale for Depression, and 2 of the Katz Adjustment Scales. Significant improvement in symptom severity was demonstrated in the nylidrin group as compared to the placebo group. There were no abnormalities of clinical significance in the safety measurements and few side effects were reported.


Prospective use of saliva lithium determinations to monitor lithium therapy.

A mathematical equation (y = 2.27x -0.45) relating saliva (y) to plasma (x) lithium concentrations was previously determined by one of the authors. A prospective study is reported here in which the correlation coefficient between the predicted and the observed plasma concentrations in a new group of 25 patients was r = 0.89 (p less than 0.001). The study demonstrates that saliva lithium determinations can be safely used to monitor lithium therapy.


Nuclear magnetic relaxation study on the interaction of glycyl-L-tyrosine with manganese-carboxypeptidase A in solution.

The spin-lattice and spin-spin relaxation rates were measured of the Gly C alpha and Tyr aryl protons of glycyl-L-tyrosine (Gly-Tyr) bound to manganese(II)-substituted carboxypeptidase A (MnCPA) in aqueous solution. The temperature and frequency dependences of the relaxation rates were analyzed using the Solomon-Bloembergen-Morgan equations. The binding modes of MnCPA with Gly-Tyr in solution are different from that of ZnCPA in crystals. 1. Mn(II)-coordinated water of MnCPA is not excluded by the binding of Gly-Tyr substrate molecules. 2. The Gly carbonyl group does not coordinate tightly to the metal ion of MnCPA. The Gly C alpha protons of Gly-Tyr in the productive binding site are appreciably mobile. 3. A non-productive loose binding of another Gly-Tyr molecule is suggested by simulation of the temperature and frequency dependences of the proton relaxation rates.


Partial purification and properties of L-2,4-diaminobutyric acid activating enzyme from a polymyxin E producing organism.

An L-2,4-diaminobutyric acid activating enzyme was found in crude extracts of Aerobacillus polyaerogenes, which produces polymyxin E1 and E2. The enzyme was partially purified by sonication of the cells, followed by ultracentrifugation, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. In addition to L-2,4-diaminobutyric acid, the enzyme activated L-leucine and L-threonine, which are constituent amino acids of polymyxin E. All three amino acids were bound to the enzyme as thioesters. These results suggest that polymyxin is synthesized by a multienzyme thiotemplate mechanism, in the same way as gramicidin S, tyrocidines, bacitracins, and gramicidin A.


Synthesis of procollagen by dental pulp cells from incisor in vitro.

Fibroblast-like cells were isolated from dental pulp of rabbit incisor, and were shown to synthesize and secrete procollagen. Analysis of the secreted procollagen and their CNBr peptides indicated that the prominent form was type I procollagen. Our results also suggested the presence of type III procollagen as a minor component synthesized by pulp cells in vitro.


Primary structure of the membrane-binding segment of rabbit cytochrome b5.

The primary structure of the membrane-binding segment of rabbit cytochrome b5 has been determined. This segment, prepared by trypsin digestion of the intact protein, consists of 43 amino acid residues and corresponds to the COOH-terminal end (residues 91-133) of the parent molecule. Deduction of the primary structure was based on automated sequence analysis of the whole segment as well as manual and dansyl-Edman degradations of peptide fragments produced by CNBr cleavage and partial acid hydrolysis. The sequence obtained is: Leu-Ser-Lys-Pro-Met-Glu-Thr-Leu-Ile-Thr-Thr-Val-Asn-Ser-Asn-Ser-Ser-Trp-Trp-Thr-Asn-Trp-Val-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Ile-Val-Ala-Leu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Asp-Asp. This sequence is 63 to 81% homologous with respect to those determined for the membrane-binding segments of equine, porcine and bovine cytochrome b5. The interaction of this segment with phospholipid bilayer membranes is discussed, and a prediction of its secondary structure is also presented.


Calcium-induced calcium release from fragmented sarcoplasmic reticulum.

A new method is introduced which allows the study of calcium-induced calcium release from fragmented sarcoplasmic reticulum. Results obtained with this method are in agreement with those obtained by previous investigators using skinned muscle fiber. It was also found that anesthetic drugs and alcohol increased the calcium- and caffeine-induced calcium release from the sarcoplasmic reticulum.


Restoration of the excitability of squid giant axon by tubulin-tyrosine ligase and microtubule proteins.

Membrane excitability of the squid giant axon was destroyed by perfusing with a medium containing 0.2 mM Ca ions that depolymerize microtubules. Restoration of the membrane excitability was achieved by perfusing the axon with a medium that contained purified tubulin-tyrosine ligase, tubulin, ATP, Mg2+, K+, cyclic AMP, and 300 K Protein.


Effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase of rat.

The effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200 mg per 100 g body weight. Catalase activity decreased to about 35% of that of the control at 42-48 h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96 h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degradation (kd) decreased to about 50% of the control.


Fluorescence studies on lipoamide dehydrogenases of pig heart. I. Conformational dynamics of enzyme.

The dynamic structures of two major forms (LD(I) and LD(II) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively. In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5. Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM. When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD(I) and LD(II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the sement of LD(I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD(II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD(I) and LD(II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.


The mechanism of the riboflavin sensitized photodestruction of mitomycin C.

A detailed in vitro study was made of the riboflavin sensitized photodestruction of mitomycin C. The dependences of the quantum yield in the system were examined on the introduction of various amounts of quenchers, such as halogen ion, paramagnetic ion, 1,4-diazabicyclo[2,2,2]octane, 2,6-di-tert-butyl-p-cresol, and p-hydroquinone. The results are consistent with a mechanism involving oxygen molecule in the excited singlet state as the photochemically reactive species. The rate constant of the reaction between the excited singlet oxygen and mitomycin C was calculated to be 8.9 x 10(9) M-1.S-1.


Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.


Isolation and characterization of two ferredoxin-NADP+ reductases from Spirulina platensis.

Two ferredoxin-NADP+ reductases (FNRs I and II) [EC 1.6.7.1] were purified from a blue-green alga, Spirulina platensis, by (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 chromatography. FNRs I and II were both FAD-containing enzymes with molecular weights of 33,000, and could photochemically reduce NADP+ to the same extent in the presence of S. platensis ferredoxin, using FNR-depleted membrane fragments of S. platensis. They had similar physical and enzymatic properties, except for chemical properties such as the amino (N)-terminal sequences and the patterns of their peptide maps. The significance of the presence of two FNRs in S. platensis as as of the multiple forms found in other organisms is discussed.


Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.


Cellular processing of pre-proparathyroid hormone involves rapid hydrolysis of the leader sequence.

The cellular synthesis of parathyroid hormone (PTH) involves two consecutive cleavages of NH2-terminal peptide sequences from a larger precursor, pre-proparathyroid hormone (Pre-ProPTH). The initial cleavage consists of the removal of an NH2-terminal leader sequence either during or shortly after biosynthesis of the polypeptide chain is complete. To determine the fate of the cleaved leader sequence, we prepared, by chemical synthesis, a peptide based on the known structure of the leader sequence of pre-proparathyroid hormone and used this peptide labeled with 125iodine as a marker to monitor the recovery of the putative cellular leader peptide during extraction and electrophoresis of [35S]methionine-labeled proteins from pulse-labeled parathyroid gland slices. Under conditions in which the recovery of the synthetic leader peptide was 50 to 70%, we found no detectable 35S-labeled product in the region of sodium dodecyl sulfate gels where the synthetic peptide migrates. In view of the known methionine content of pre-proparathyroid hormone and proparathyroid hormone (ProPTH), it would have been possible to detect endogenously labeled leader peptide if present in amounts equal to 0.05% of the amount of labeled ProPTH present in the tissues. These observations indicate that the cellular conversion of Pre-ProPTH to ProPTH involves a rapid hydrolysis of the leader peptide either during or immediately after its removal from the precursor.


Resolution of the charge forms and amino acid sequence and location of a tryptic glycopeptide in rat alpha-lactalbumin.

Three charge forms of rat alpha-lactalbumin were separated by ion exchange chromatography on DEAE-cellulose. The amino acid composition of each form was similar but they differed in carbohydrate composition. Each form contained a tryptic glycopeptide having a common polypeptide and heteropolysaccharide unit. The tryptic glycopeptide was sequenced and positioned in rat alpha-lactalbumin, which was partially sequenced from residues 1 to 50. The carbohydrate attachment site was at Asn45. Secondary structure calculations predicted that Asn45 is in a beta bend conformation whereas Asn45 in bovine alpha-lactalbumin, a poorly glycosylated protein, is not in a bend conformation.


Isolation and partial characterization of a new soluble b-type cytochrome (b9) from rat liver.

A new soluble cytochrome, designated as cytochrome b9, was purified to apparent homogeneity from rat liver. The absorption maximum of the oxidized (the native form) cytochrome b9 at room temperature was 413 nm. The dithionite-reduced cytochrome b9 had absorption maxima at 556, 527, and 423 nm. The prosthetic group of cytochrome b9 was identified as protoheme IX. From gel filtration experiments, the molecular weight of cytochrome b9 was estimated to be 125,000. Polyacrylamide gel electrophoresis experiments in the presence of sodium dodecyl sulfate showed that the molecular weight of its subunit was 61,000. The native form of cytochrome b9 was thus a dimer. The amount of heme/mol of dimer was 3.3 mol. Cytochrome b9 was autoxidizable and did not bind CO, 2.2 mM cyanide, or 2.2 mM azide. On the basis of its molecular weight of 125,000, the millimolar extinction coefficients of dimeric cytochrome b9 at 280 and 413 nm were 384 and 380, respectively. The absorbance at 280 nm/mg cytochrome b9 was 3.1. Cytochromes b9 and H-450 (I.-C. Kim and W.C. Deal (1976) Biochemistry 15, 4925-4930) are the only b-type, soluble cytochromes which have been isolated from mammalian liver; they are not found in tissues of heart, lung, kidney, and brain. The biological function of cytochrome b9 was not determined.


Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.


The role of the kidney in the biosynthesis of carnitine in the rat.

Intravenous administration of L-[methyl-3H]-labeled trimethyllysine to rats results in a very rapid accumulation of radioactivity by the kidneys, while the incorporation of the label into the liver occurs at approximately 1% of this rate when calculated per g of wet tissue. The kidneys convert a substantial portion of the trimethyllysine taken up to butyrobetaine and to beta-hydroxytrimethyllysine, a precursor of butyrobetaine, but fail to synthesize carnitine. Significant amounts of radioactivity are recovered in both carnitine and butyrobetaine of hepatic tissue after longer time periods, while the level of labeled trimethyllysine in this organ remains very low. Bilateral nephrectomy results in a marked decrease in the incorporation of label into the liver. These results indicate that in rats, the initial conversion of trimethyllysine to butyrobetaine occurs predominantly in kidney and that the liver capacity for this transformation is considerably smaller than its capacity to synthesize carnitine from butyrobetaine.


Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides.

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.


Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.


Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.


Kinetics of adhesion and de-adhesion of Chlamydomonas gametes.

In medium with low nitrogen content, vegetative strains of the unicellular biflagellate alga Chlamydomonas reinhardi form gametes. Mating type plus (mt+) and mating type minus (mt-) gametes adhere via their flagella to give aggregates in which the gametes eventually fuse to form zygotes. A quantitative assay has now been developed which measures aggregation and fusion by use of a Coulter electronic particle counter to determine loss of single gametes as they form aggregates in suspension. Determination of the rate and extent of cell fusion by microscopy agrees with the results obtained with the more rapid and convenient Coulter counter assay. By use of the assay it was found that aggregation and fusion occur at the same rate and to the same extent at 12 degrees C and 25 degrees C. Flagella from one of the mating types can specifically substitute for the corresponding live gametes; more than 70% of the gametes were aggregated and the extent of aggregation was proportional to the number of flagella added, until the ratio of cells to flagella exceeded 2. At 22 degrees C, in the flagella/gamete mixtures, adhesion was complete in less than 5 min, but at 5 to 10 min, gametes began to de-adhere from the clusters and, depending on the number of flagella added, essentially all of the gametes detached from the aggregates in 10 to 50 min. The gametes in such mixtures were fully competent to aggregate again, whereas the flagella recovered from such mixtures were shown by use of a radioactive flagella-binding assay to be inactive with fresh gametes. Inactivation of the flagella was temperature-dependent, was not catalyzed by soluble factors, and required adhesion of flagella to gametes of the opposite mating type. The potential physiological functions of the de-adhesion process are discussed.


Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.


Reaction of fungal amine oxidase with beta-bromoethylamine.

beta-Br-ethylamine is both a substrate and an irreversible inhibitor of amine oxidase from Aspergillus niger. The enzyme catalyzes the nonoxidative elimination of HBr from beta-Br-ethylamine to form acetaldehyde. beta-Br-ethylamine meets several criteria for an irreversible substrate analog or suicide inhibitor. 1) It inactivates the oxidized enzyme, but not the reduced enzyme. 2) The Michaelis constant for beta-Br-ethylamine in the elimination reaction showed a similar magnitude to that of the related constant found when the haloamine acted as an inhibitor. 3) The enzyme was protected from the inactivation by the co-existence of the substrate. 4) Inactivation with beta-Br-[14C]ethylamine resulted in the incorporation of radioactivity corresponding to 1 mol of the label/mol of the monomeric unit of the enzyme and a decrease of 1 mol of the -SH group. 5) Inactivation was accompanied by the formation of a new absorption peak at 320 nm which was bleached by addition of NaBH4.


The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.


Reaction of the histidines of prolactin with ethoxyformic anhydride. A binding site modification.

The seven histidines of bovine prolactin were modified with ethoxyformic anhydride and two classes of reactivity were apparent: 5 histidines were in the more reactive class (k = 0.097 min-1) and 2 histidines were less reactive (k = 0.011 min-1). The activity of the modified prolactins was determined by measuring their ability to bind to prolactin receptors from rabbit mammary glands. This assay showed that prolactin was fully active when 0 to 5 histidines were modified. If all 7 residues were modified, the hormone was unable to bind to its receptor. Circular dichroism studies indicated no significant differences in conformation for prolactins which had 2 to 7 histidines modified. Modification of human growth hormone and human placental lactogen with ethoxyformic anhydride resulted in a loss of the ability of these lactogenic hormones to bind to the prolactin receptor. For all three hormones, essentially full activity was recovered when the modifying group was removed by treatment with hydroxylamine. Sequence comparisons indicate that only 2 of the 3 growth hormone histidines and 2 of 7 placental lactogen histidines were homologous with histidines in bovine prolactin and that, in each case, they correspond to His-27 and His-30 in bovine prolactin. It is postulated that these residues serve to identify a portion of the binding domain of bovine prolactin.


Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship.

Two antibacterial proteins from rabbit polymorphonuclear leukocytes, a potent bactericidal cationic protein that increases the envelope permeability of susceptible gram-negative bacteria and a phospholipase A2, have been purified to near homogeneity by ion exchange, gel filtration, and hydrophobic interaction chromatography. The apparently noncatalytic bactericidal/permeability-increasing protein has an approximate molecular weight of 50,000 and is isoelectric at pH 9.5 to 10.0. The molecular properties, including amino acid composition, and the antibacterial potency and specificity of this rabbit leukocyte protein and of the bactericidal/permeability-increasing protein from human granulocytes that we have recently purified (J. Biol. Chem. 253, 2664-2672, 1978) are closely similar. Both proteins kill several strains of Escherichia coli and Salmonella typhimurium. Rough strains are more sensitive than smooth strains. All gram-positive bacterial species tested are insensitive to high concentrations of either rabbit or human protein. The phospholipase A2, purified by hydrophobic interaction chromatography on phenyl-Sepharose, ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 14,000 and had a specific enzymatic activity comparable to that of purified phospholipases A2 from other sources. Separation of the phospholipase A2 from the bactericidal/permeability-increasing protein has no noticeable effect on the bactericidal and permeability-increasing activities of the purified bactericidal protein, but removes the ability of the phospholipase A2 to hydrolyze the phospholipids of intact Escherichia coli. Upon recombination of the phospholipase A2 with the bactericidal/permeability-increasing protein, the phospholipase A2 regains its activity toward the phospholipids of intact E. coli suggesting that these two antibacterial leukocyte proteins act in concert.


Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.


Pathway-dependent reconstitution of chromatin structure from separated constituents.

Chicken reticulocyte chromatin can be reassembled from its separated constituents, viz. DNA, H1 plus H5, core histones, and non-histone proteins, to yield a product resembling the native starting material by a series of structural criteria. In particular, it possesses nucleosomes separated by spacer regions; the particles contain DNA with a unit length of approximately 200 base pairs. The recovery of the correctly reassembled product depends critically on the annealing conditions: the components are initially mixed in 2 M NaCl and 5 M urea, and it seems to be important to remove urea at a relatively high salt concentration. The results suggest that the characteristic chromatin structure is formed only when core histones bind to DNA in their native conformation and are followed by the addition of H1 and H5 to the spacer regions.


Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.

A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.


Nucleotide sequence of nucleolar U3B RNA.

U3A, U3B, and U3C are three distinct molecular weight nucleolar RNAs present in Novikoff hepatoma ascites cells. The primary nucleotide sequence of U3B, the most prominent of these U3 species, was determined. Purified U3B RNA was subjected to various enzymatic digestion procedures, including digests of 32P-labeled U3B RNA, RNA ligase, and polynucleotide kinase labeling, for determination of its primary sequence which is: (formula: see text). The 5'-terminus of the RNA has a "cap" and localized purine-rich regions were found near the 3'-terminus, which have been incorporated into a hydrogen-bonded region in a proposed secondary structure of the molecule.