Recovery of prolactin from acetone-dried human pituitary glands.

An improved method is described for the purification of prolactin from the same batch of acetone-dried pituitary glands which is used for the isolation of other anterior pituitary hormones. The residue obtained after the extraction of the glycoprotein fraction from the pituitary acetone powder was extracted for growth hormone. The supernatant after the precipitation of growth hormone at 50% ethanol concentration was adjusted to 85% in ethanol. To the supernatant obtained from that step an equal volume of acetone was added to precipitate the 'PRL fraction.' This fraction was further purified by gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose and isoelectric focussing. A yield of 23 mg prolactin, 37 U/mg, was obtained per 1000 g pituitary acetone powder.


Placental steriodogenesis in the rat: comparison of normal and giant placentae.

Placental hypertrophy was induced in pregnant rats by daily treatment with estrone (0.5 microng) and progesterone (4 mg) from day 3 through 19 combined with ovariectomy on day 12 of pregnancy. The ratio of basal zone to whole placenta was 47% by weight in these enlarged placentae on day 20 of pregnancy but only 34% in normal placentae. In each case, the basal zone tissue was homogenized and the 10,000 x g supernatant fraction was used for in vitro incubation with [7alpha-3H]pregnenolone as the added substrate. The normal placental tissue synthesized more progesterone and testosterone but less androstenedione and 5alpha-pregnane-3,20-dione than the giant placentae. Utilization of the pregnenolone substrate by the giant placentae was subnormal. Addition of 200 IU of human chorionic gonadotropin (hCG) in vitro to normal basal zone placentae sharply increased the production of progesterone, 17alpha-hydroxyprogesterone, androstenedione and 5alpha-pregnane-3,20-dione. The giant placentage failed to respond to hCG. Thus, steroid-induced hypertrophied placentae in rats do not participate in a compensatory mechanism for steroid hormone production toward pregnancy maintenance.


Studies on the mechanism by which thyroid hormones enhance alpha-lactalbumin activity in explants from mouse mammary glands.

Addition of 3,5,3'-triiodo-L-thyronine to cultures of mammary gland explants in serumfree medium containing insulin, hydrocortisone and prolactin results in a 3 to 5-fold increase in the activity of the milk-protein alpha-lactalbumin over that seen in the presence of the latter three hormones alone. The thyroid hormone does not act by substituting for any of the other hormones. It need not be present throughout the culture period but can act if added along with prolactin after insulin and hydrocortisone have induced formation of the rough endoplasmic reticulum. Delayed addition of the thyroid hormone also results in further stimulation of cells already responding maximally to insulin, hydrocortisone and prolactin. These effects of triiodothyronine are not blocked by progesterone at 1 microng per ml. They are, however, blocked by the addition of inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis, suggesting that the thyroid hormone increases the synthesis of the alpha-lactalbumin molecule itself. The thyroid hormone appears to act by altering the responsiveness of the mammary gland explants to prolactin, but not to insulin or hydrocortisone. In the presence of 10-9M triiodothyronine, enhanced alpha-lactalbumin activity is consistently obtained at prolactin concentrations as low as 4.5 x 10(-12)M whereas, in the absence of the thyroid hormone, ten times more prolactin (4.5 x 10(-11)M) is needed to obtain an increase in alpha-lactalbumin activity.


A new method for the assessment of thyroxine metabolism in the rat.

A new method for assessing thyroxine (T4) metabolism in the rat has been devised and the results that it yields have been compared to those obtained by other methods for assessing peripheral T4 metabolism. The new method uses a simple analysis of the kinetics of exogenous T4 metabolism in rats chronically treated with daily injections of exogenous labeled T4 to determine T4 clearance rates via both fecal and deiodinative pathways. Fecal clearance rates of endogenously labeled hormone measured in rats brought into approximate 125I/127I specific activity equilibrium by administration of iodide of know specific activity in the drinking water were nearly the same as those determined with this new method. Furthermore, total T4 clearance rate determined with the new method agreed well with that measured by the standard single injected technique utilizing radioactive T4. These results indicate that quantitation of the clearance rate of endogenous T4 via not only the fecal, but also the deiodinative, pathway for T4 disposal can be achieved with this new method. In rats made hyperthyroid with exogenous T4, clearance rates of T4 via both deiodinative and fecal pathways were markedly increased. This result supports the concept that hyperthyroidism increases the activity of the tissue mechanisms for metabolizing T4.


The effects of gamma-aminobutyric acid on prolactin and gonadotropin secretion in the unanesthetized rat.

The effects of the intraventricular injection of gamma-aminobutyric acid (GABA) on pituitary secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL) were examined in Na-pentobarbital (PB)-anesthetized and unanesthetized male rats. GABA (5 or 10 millimicronmol) injected into the lateral ventricle of PB anesthetized rats resulted in significant elevations in plasma concentrations of LH and PRL. When administered via a lateral ventricle cannula to unanesthetized rats, GABA (1, 5, or 10 millimicronmol) caused a significant increase in plasma PRL levels but had no effect on plasm LH concentrations. Plasma FSH was uninfluenced by GABA in all animals studied. The data indicate that PB anesthesia can alter the hypothalamic pituitary axis to a possible neurotransmitter agent.


Rise of plasma corticosterone concentrations in rats immediately before and after birth and in fetal rats after the ligation of maternal uterine blood vessels or of the umbilical cord.

Just before normal vaginal delivery the plasma corticosterone concentration (PCC) in fetal rats increased markedly compared with that on day 22 of gestation. The PCC was elevated 2 h after birth and then declined until 12 h after birth, when it approached the level found on day 22 of gestation. Similarly, in premature newborn rats obtained by cesarean delivery on days 21 and 22 of gestation, the PCC increased 2 to 4 h after delivery and then declined. The PCC in 22-day-old fetal rats, 30 min after ligation of the maternal blood vessels running along a uterine horn, rose markedly compared with that in fetuses in the contralateral intact horn. A similar increase in PCC was not observed in either decapitated or encephalectomized fetuses, but observed in fetuses whose umbilical cords were ligated. The rise in fetal PCC after ligation of uterine blood vessels and of umbilical cords was considerably higher than that observed after various stresses reported by other investigators and also higher than that found 60 min after maternal adrenalectomy. The rise in PCC just before and just after birth and after the ligation should not be considered as the result of stress, parturition or ligation, but may be a result of the response to other factors.


Characterization of abnormal thyroglobulin in a transplantable rat thyroid tumor.

The solube iodoproteins in a transplantable rat throid tumor (Wollman Line 1-8) were studied after in vivo labeling with 125 I and were partially purified by affinity chromatography on anti-thyroglobulin-AGAROSE. A major fraction ('Peak A') was excluded from gels of large pore size, but had a low sedimentation rate (approximately8S) and did not appear to contain aggregates. It had a high density (approximately1.4) which was possibly due to a high content of carbohydrate, since treatment with a crude glycosidase mixture lowered the density to approximately1.3. A second fraction ('Peak B') had a similar sedimentation coefficient (6-9S) but penetrated the same gels and had a lower density (approximately 1.3). Both proteins formed soluble complexes with antibodies against normal rat thyroglobulin, and had other properties somewhat similar to those of thyroglobulin. After hydrolysis, mono- and diiodotyrosine were the only iodoamino acids liberated. These iodoproteins appears to represent abnormal forms of thyroglobulin synthesized by the tumor.


Regulation of the cell cycle by diethylstilbestrol and progesterone in cultured endometrial cells.

Cultures of primary explanted rabbit endometrium epithelial cells were found to be a mixture of dividing or cycling (20%) and non-dividing or GO (80%) cells. The addition of diethylstilbestrol had the effect of switching most of the GO cells into the cell cycle and to shorten the length of the cell cycle through a shortening of G1 and S. Progesterone had the opposite effects.


Plasma testosterone during pregnancy in the mouse.

Levels of plasma testosterone (T) were studied throughout pregnancy and on the day of parturition in selected strains of mice. A dramatic midpregnancy increase in androgen occurred in both strains examined (peak on day 9). A second increase in plasma T was found during the latter half of gestation (days 14-17), at which time plasma estradiol levels were elevated.


Amino-acid sequences of heme-linked, histidine-containing peptides of five peroxidases from horseradish and turnip.

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences nearthe peroxidase heme prostetic group. In the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heme, and 34 residues around the probably distally located histidine, and compare them with the histidine-containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3, and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physio-chemical and enzymic properties. Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site.


The trypsin and chymotrypsin inhibitors in chick pears (Cicer arietinum L). The relationships among the six isoinhibitors.

The six isoinhibitors of trypsin and chymotrypsin which were previously purified from a crude extract of chick peas were further studied with the intention of establishing the relationships among them.  Exposure of each fraction to active, matrix-bound trypsin resulted in a decrease of the specific inhibitory activities towards trypsin of fractions P-1, P-3 and P-5 while those of P-4 and P-6 were increased. Each of these fractions was separated into several sub-fractions after chromatography on a DEAE-Sephadex A-25 column under conditions identical to those used previously for their purification. The elution positions, specific inhibitory activities towards trypsin and electrophoretic patterns in polyacrylamide gels of the various sub-fractions were determined...


A polyvalent proteinase inhibitor from horse-blood-leucocyte cytosol. Isolation, purification and some molecular parameters.

Cytosol of horse blood polymorphonuclear leucocytes contains an inhibitor active against neutral proteinases from the granules of these cells and against chymotrypsin, elastases I and II from pig pancreas, but not against trypsin. A method has been elaborated to isolate and purify this inhibitor by means of salting out with ammonium sulphate (45---70% saturation), followed by chromatography and rechromatography on a column of DEAE-Sephadex A-50. The preparation obtained is homogeneous during polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecylsulphate after reduction with 2-mercaptoethanol. It is a thermolabile protein soluble at its isoelectric point (pH 5.38) and having a molecular weight of 35200 but aggregating in solutions of low ionic strength and irreversibly precipitating during dialysis at pH 4.0. The results obtained show the similarity of this inhibitor isolated from horse blood leucocyte cytosol to other inhibitors isolated from human or pig blood leucocytes.


Structure and synthesis of a lipid-containing bacteriophage. Effects of lipids containing cis or trans fatty acids on the reconstitution of bacteriophage PM2.

Infectious PM2 virus paticles could be reconstituted in vitro from a mixture of nucleocapsid, phospholipids containing cis fatty acids, and proteins I and II. The presence or absence of acyl phosphatidylglycerol, a minor lipid component of thevirion, did not affect the reconstitution of infectious particles, even though it was incorporated into the particles when present. When phosphatidylglycerol was completely replaced by acyl phosphatidylglycerol in the reconstitution mixture, no infections particles were formed. Lipids containing either cis or trans fatty acids were also used for reconstitution in vitro of the lipid-containing bacteriophage PM2. Regardless of the ratio of phosphatidlyglycerol to phospatidylethanolamine in the reconstitution mixture, infectious particles were formed and had almost the same phospholipid composition when lipids containing cis-palmitoleic acid were used; no infectious particles were obtained when lipids containing trans-palmitoleic acid were used. In the latter case, virus-like particles were, however, formed. Reconstituted particles containing cis fatty acids were infectious when tested on wild type Pseudomonas BAL-31 as well as on the unsaturated fatty acid auxotroph grown in the presence of either cis or trans-palmitoleic acid. Reconstituted particles containing trans fatty acids were not infectious on any of these cells. When trans fatty acids as well as cis fatty acids were present in the reconstitution mixture, then there was a lower yield of infectious particles. Particles with either cis or trans fatty acids had all four viral proteins and adsorbed to BAL-31 host cells in a specific manner.


DNA polymerases from intracisternal A-type particles on the mouse plasmacytoma MPC 11.

Intracisternal A particles from mouse plasma cell tumors were isolated from microsomal vesiclels by detergent treatment and separated in linear sucrose gradients. Four peaks of DNA polymerase activities banding at densities of 1.30, 1.24, 1.20--1.22 and 1.13 g/cm3 were observed assaying on poly(rA).oligo(dT). Solubolized DNA polymerases of the 1.30 g/cm3...


Multiple forms of DNA-dependent RNA polymerase I from immature chick liver. Selective effect of 17beta-estradiol.

1. We found that a single injection of 17beta-estradiol into immature chicks resulted within 24 h in a 2-fold increase of the transcripitional capacity of liver nuclei which involved both endogenous RNA polymerase I and II actvities. Similarly, RNA polymerase activity I of purified nucleoli was also doubled. 2. During purification of RNA polymerase I from controls and treated chicks the difference in activity was almost completely lost. 3. RNA polymerase I could be resolved into two forms, IA and IB, on CM-Spehadex. Form IA increased and form IB decreased after estrogen treatment while the sum of the two remained constant. Form IA sedimented more slowly than form IB in glycerol gradients and was predominant in purified nucleoli. 4. These observations suggest that there may be an equilibrium between the two forms of RNA polymerase I which may change in order to produce the translational shift-up which occurs after estrogen treatment.


Membrane-dependent cleavage of the human placental lactogen precursor to its native form in ascites cell-free extracts.

Messenger RNA derived from term placenta directs the synthesis of a precursor to human placental lactogen (prelactogen, molecular weight 25000) in an ascites cell-free system containing ribosome free supernatant and preincubated purified ribosomes. The processing of prelactogen to native lactogen (molecular weight 22200) was only observed when a microsomal membrane preparation was added prior to the synthesis of complete protein, i.e. before release. Placental mRNA directed the synthesis of prelactogen in asystem containing free polysomes, whereas in a comparable system containing membrane-bound polysomes both prelactogen and lactogen were synthesized. The prelactogen synthesized in the latter system could be cleaved by the addition of membranes at the start of incubation. Preprotein cleavage activity was inhibited 100% by 0.04% Tritonx-100, while protein synthesis was inhibited only about 30%. Using Triton to block cleavage specifically at intervals after mRNA and membrane additions, it was determined that the overall cleavage reaction required about 15 min. When the ascites system was incubated with charged initiator [35S]methionyl-tRNAfMet prelactogen was formed. The labeled prelactogen was processed when membranes were added after 60 min of incubation. The results indicate that prelactogen is the primary gene product, and cleavage activity is apparently associated only with the membrane-bound ribosomal fraction of the cell.


Specificity profiles of the membrane-bound gamma-D-glutamyl-(L)meso-diaminopimelateendopeptidase and LD-carboxypeptidase from Bacillus sphaericus 9602.

Immunological studies on the carboxypeptidase Y mutant prel-1 of Saccharomyces cerevisiae revealed the origin of mutation in the structural gene of carboxypeptidase Y. The absence of carboxypeptidase Y has no effect on growth, even after drastic changes of growth conditions...


Secondary and conformational specificities of trypsin and chymotrypsin.

Specific and non-specific trypsin substrates of known structure have been examined for common features. This analysis suggests that trypsin has a specificity for a particular conformation near the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed helix at the third and fourth (P3 and P4) amino acid positions toward the amino terminus from the scissile bond which I denote as a conformational specificity. This conformation is a bent left-handed show a high frequency of proline and glycine at these positions consistent with the left-handed helical conformation. This apparent secondary specificity for a particular substrate residue other than that at the primary position is not related to the nature of the residues at the third and fourth positions. Rather, these residues determine the bend of left-handed helix which has the effect of exposing main chain hydrogen-bonding groups of the substrate peptide chain to hydrogen-bonding groups on the enzyme. Thus, the secondary specificity of trypsin is not sequence-specific, but is for peptide main chain in the third and fourth positions and is determined by the tertiary structure of the substrate. This hypothesis for conformational and secondary specificity in trypsin can be extended to chymotrypsin. It also provides a means for the regulation of certain processes in vivo catalyzed by other proteases.


[Methionyl-tRNA synthetase from wheat embryo: dissociation into subunits (author's transl)].

Wheat -embryo methionyl-tRNA synthetase is a dimeric protein of beta2 structure. When highly diluted, it loses the capacity to catalyze ATP-[32P]PPi exchange and to aminoacylate tRNAMet: at low enzymatic concentrations the rates of formation of[32P]ATP and [14C]methionyl-tRNAMet are lower than those predicatedby extrapolating the rates determined at higher enzyme concentrations. The difference between observed and expected rates becomes greater with decreasing enzyme concentration. Filtration of purified, dilute enzyme preparations on Sephadex G-200 results in the separation of dimer and monomer fractions. The proportion of monomer present increases with increasing pre-incubation times before the assay and demonstrates an equilibrium between active dimers and being shifted towards the production of monomers. Datapreviously gathered for Escherichia coli prolyl-tRNA synthetase and for bovine-pancreatic tryptophanyl-tRNA synthetase, coupled with the present results, suggests that the dissociation of dimeric synthetases may be a general phenomenon in eukaryotes as well as in prokaryotes. The number of sub-units and the dissociation constant were obtained at equilibrium, according to relations adapted to the case of oligomeric enzymes (KD congruent to 13 nM at 25 degrees C and pH 7.5). Rate constants were determined by kinetic studies of the attainment of equilibrium. The rate constant k1 for monomolecular dissociation was determined to be 1.85- 10-3 s-1 and k2 for the bimolecular association to be 0.145 - 106 M-1 s-1. The KD calculated from k1 and k2 was coherent with the experimentally determined value, at equilibrium. The sub-unit interactions, which involve only a small quantity of energy (delta G degree congruent to + 11 kcal mol-1; +45 kJ mol-1) at 25 degrees C, depend on the ionic environment of the medium and the presence of substrates. Alkaline pH favors monomer production, while the presence of methionine, (Mg-ATP)2- and tRNAMet protect the synthetase from dissociation. 2-mercaptoethanol and dithioerythritol prevent only slightly the loss of activity. Bovine serum albumin, however, protects the enzyme from dissociation under dilute conditions.


[Induction of malformations by N-methyl-N-nitrosourea (MNU) (author's transl))].

After observing multiple malformations in a stillborn infant whose mother was in contact with MNU during pregnancy, the inhalation of evaporated MNU solution or diazomethane was regarded as a possible cause. An attempt was made to reproduce the same conditions in experiments with rats. The experiments were carried out with 46 pregnant rats yielding 370 fetuses. With the aid of an atomizer a solution of 50 mg MNU was atomized in a container of 12 liters. In each test up to 3 rats were exposed for 30 min (group A1). In other groups (B1--3) rats placed into the same container breathed diazomethane which was produced by dropping NaOH on MNU. During this experiment, rats were apathetic and their respiration was accelerated. In group C1--9, MNU was injected intraveneously or intraperitoneally. Animals which had no contact neither with complete MNU nor with its decomposition products served as control (group D1). Rats were killed at the 21st day of pregnancy. After laparotomy, fetuses were weighed and fixed in Bouin's solution or in ethanol. Fetuses were examined macroscopically. Afterwards the head and the trunk were dissected. In a number of cases the skeleton was stained with alizarin red S. Malformations that affected the skeleton and the central nervous system could be found only in those experiments in which rats were exposed to the complete teratogenic agent. By means of external inspection microcephalia, micrognathia, cleft palate, micromelia, syn- and polydactylia, lordosis and skoliosis of the spinal column as well as deviations of the extremities were observed. Above this, by bone preparation by alizarin red S malformations of the skull (micrognathia, aplasia of the zygomatic bone) hypoplasia and deformations of the clavicula, scapula, ribs as well as of bones of the extremities were found. MNU led to complex malformations in the central nervous system comprising microencephalia, hydrocephalus as well as defects of the septum pellucidum and the corpus callosum. Frequency and spectrum of malformations depended on the dosage and the age of pregnancy. As the embryotoxic activity was relatively low at day 13 and as malformations were observed in all cases, this day proved to be most effective one. In contrast to the application of MNU, after inhalation of diazomethane malformations did not occur. However, as a toxic effect a significant increase in dead fetuses and a reduction of the body weight was registered. After inhalation of atomized MNU solution, neither malformations nor toxic injuries could be observed. The experiments do not support the hypothesis, that anomalies found in the stillborn infant are caused by mother's contact with MNU. As demonstrated, in most cases it is very difficult to prove any interrelationship between a chemical agent and the occurrence of malformations in man. Therefore, similar observations should be published even if the connection cannot be ascertained exactly, for only by an accumulation of related cases a positive correlation can be verified.


[Electron microscopic investigations on early stages of experimental heart tumours in rats (author's transl)].

The views on the histogenesis of experimental heart tumours in rats are controversal. Up to now electron microscopic investigations on early tumour stages are lacking. In this study ultrastructural findings of 4 early stages of heart neoplasms in the left ventricle of BD-IX rats are reported. Material and methods. BD-IX rats received repeated intravenous injections of methylnitrosourea in a dosage of 20 mg/kg body weight. After averagely 9 months the animals were killed by ether anesthesia. Sections of the heart were fixed in solution according to Karlsson and Schultz (1965). In order to discover early stages of tumours light microscopic examinations were carried out on heart sections. Afterwards the small tumour-containing tissue samples were postfixed in 1% OsO4, embedded in durcupan, and double contrasted with uranyl acetate and lead citrate. Results. Light microscopy (semithin sections): Beneath an endothelium-like cell layer an area of parallel to each other arranged spindle-shaped tumour cells with usually hyperchromatic nuclei was found. Some cells were larger and contained pale nuclei. Electron microscopy: The spindle-shaped neoplastic cells with irregular nuclei are loosely arranged. Most of the cells were surrounded by a basement membrane. In the extracellular space abundant thin cell processes and collagen fibers were demonstrable. The tumours contained characteristic bundles of cell processes as well as single processes which enveloped a virtual point of the space. Some large and widely undifferentiated tumour cells had stump-like thick processes. In the area of trabecular muscle neoplastic cells of an apparently mesenchymal origin were present. The ultrastructural findings are discussed regarding the histogenesis of the small heart tumours in the rats. They probably developed from neoplastic Schwann cells but an additional mesenchymal component seems to be engaged. Further studies are necessary to clarify the significance of both components and their relations in the experimental heart tumours.


Morphology of diethylnitrosamine-induced lung tumours in Dzungarian dwarf hamsters.

After transplacental application of 30 mg/kg b.w. diethylnitrosamine 16 out of 21 Dzungarian dwarf hamsters developed lung tumours. The histological picture of these tumours is described. The neoplasms are classified as papillary, tubular, or alveolar adenomas or adenocarcinomas, respectively. Different histological architectures occur in one and the same animal or even in different parts of the same tumour. The neoplasms derive from outgrowths of the epithelium of small bronchioles. The significance of the Dzungarian dwarf hamster as a suitable tool for transplacental and postnatal cancer research is discussed.


[Experimental studies on disseminated intravascular coagulation (author's transl)].

Generalized disseminated intravascular coagulation (DIC) was produced in rats by thrombin infusion (550 NIH U thrombin/kg/h). Changes in the clotting system and morphological alterations were studied with consideration to early lesions. Furthermore, the influence of the antifibrinolytic agent Pamba and the fibrinolytic enzyme ocrase on DIC are studied. Microthrombi and degenerative alterations are increased in parenchymatous organs after thrombin infusion with Pamba is applied, they are decreased when ocrase is given. Alterations are most prominent in the lungs revealing the appearance of a "shock lung". Semiquantitative morphological examinations of lung, liver, kidney and adrenal glands were performed by counting microthrombi. There are marked differences between the test groups.


[Action of chlorphentermine on the hydrolysis of phosphatidyl choline by phospholipase A2 (author's transl)].

The anorectic drug chlorphentermine (Chlph) has been reported to cause lipoidosis-like cellular alterations in many organs, especially in lungs. A weak inflammation has been observed during the first week of daily application. After that time a pronounced foam cell production with many lamellated inclusion bodies in the cell occurs. It has been supposed that this action is caused by an inhibition of phospholipid-degradation due to an association between the drug and phospholipids. To substantiate this mechanism an in vitro study a bout the action of Chlph on the hydrolysis of phosphytidyl choline (PC) by phospholipase A2 (bee venom) was undertaken. Pur PC was obtained by column chromatography of egg lecithin. PC was used in two physical states. Handshaken liposomes are used as a model of the lamellated inclusion bodies of drug- induced phospholipidosis. The kinetic analysis was carried out on single bilayered liposomes obtained by injection of an ethanolic PC-solution into 0.16 M KCl. Purified bee venom was used as enzyme source. Usually the drug was added before the initiation of the enzyme reaction. In some cases Chlph was added after starting the hydrolysis by phospholipase for a detailed characterization of the type of interaction between Chlph and PC. The velocity of the enzyme reaction was measured by pH- stat titration and was expressed as mM H+-release per min. Two phases of Chlph-action are observed. A time limited stimulation of hydrolysis occurs immediatly after addition of the drug. The enzyme reaction is inhibited after the disappearence of this activation. This inhibition is independent of the physical state of the substrate and is very pronounced at equimolar mixtures of Chlph and PC (88 per cent inhibition in handshaken liposomes; 78 per cent inhibition in single bilayered liposomes). At inhibitor concentrations below 10 mol per cent the hydrolysis is not affected. By kinetic analysis it was found that the inhibitory action is due to an association between the inhibitor and the substrate. The Lineweaver-Burk- and Dixon-replots show a series of curves characteristic for this type of interaction (concave shape; no common intersections, situated in the 2nd quadrant). The intermediate stimulation of the substrate hydrolysis occurs only during the reaction of Chlph with PC. This is concluded from the following observations: The duration of activation is proportional to the inhibitor concentration as well as to the substrate concentration, i.e. it is proportional to the concentration of both reactants. The activation does not occur if the enzyme reaction is started some time after mixing inhibitor and substrate, i.e. after finishing the reaction. The results are discussed in relation to the in vivo action of Chlph.


[Lack of a cancerogenic effect after application of amines and amides together with nitrite in rabbits (author's transl)].

Young rabbits of both sexes received the following nitrosable compounds and sodium nitrite: Group 1:13 animals received 0.1 per cent methylurea in food and 0.1 per cent NaNO2 in drinking water. Two control groups of 6 rabbits were applied NaNO2 or methylurea, respectively. Duration of application 100 days. Group2: 20 animals received 0.2 per cent dimethylbenzylamine in drinking water and 0.15 per cent NaNO2 in food for 100 days. 10 control animals got only 0.2 per cent dimethylbenzylamine. Group 3: 20 rabbits were fed with 0.2 per cent diethylaminohydrochloride and 0.2 per cent NaNO2 added to the food. 10 controls received only 0.2 per cent of the amine. Duration of feeding 63 days.


Effect of ethanol on stimulus-induced insulin secretion and glucose tolerance. A study of mechanisms.

The effect of ethanol on stimulus-induced insulin secretion was studied, and possible mechanisms were examined in fasting unanesthetized and unrestrained rats with indwelling jugular and aortic catheters. Glucose (150 mg.) or tolbutamide (10 mg.) was given rapidly, i.v., one hour after  agavage of ethanol or saline (control). Acutely, ethanol treatment caused marked inhibition of glucose-induced insulin secretion and impaired glucose disappearance rate. Tolbutamide-induced insulin secretion was also significantly inhibited, and decline in glucose was significantly less in ethanol-treated rats. In response to ethanol, serum calcium concentration significantly declined for two hours. In another study, an ethanol metabolite, acetate (0.4 micronmole/min.) or vehicle (control) was infused for 60 minutes prior to 150 mg. glucose pulse. Acetate priming significantly potentiated glucose-induced insulin secretion and also improved glucose tolerance. It is proposed that (1) ethanol in vivo acutely induces hypocalcemia, which inhibits glucose- and tolbutamide-induced insulin secretion--which, in turn, causes glucose intolerance and prevents tolbutamide-induced hypoglycemia. (2)Acetate might be the actual petentiating influence on glucose-induced insulin secretion observed several hours after ethanol treatment.


Abnormalities in proliferation and protein synthesis in skin fibroblast cultures from patients with diabetes mellitus.

Several aspects of in-vitro cell growth and protein synthesis were assessed in cultures of skin fibroblasts from subjects with juvenile-onset diabetes mellitus (JODM) or adult-onset diabetes mellitus (AODM) and from age-matched nondiabetic controls (C). There was an inverse correlation between increasing age and both the log-phase doubling rate and saturation density at confluence in C fibroblasts. JODM and AODM cells had a reduction in both indices of cell population growth in comparison with age-matched C fibroblasts. Fibroblasts grown in the presence of 0.3 micronM hydrocortisone were stimulated to grow more rapidly and to a greater saturation density. Stimulation of cell division by hydrocortisone accentuated the abnormalities in growth of JODM and AODM fibroblasts. Total protein and collagen synthesis was measured whtn the fibroblasts had grown to confluency in medium with or without hydrocorticone. Hydrocorticone did not produce a significant change in total protein and collagen synthesis per cell by C fibroblasts. Fibroblasts from AODM had a 180 per cent increase in total protein and collagen synthesis in the presence of hydrocortisone. In contrast, total protein and collagen synthesis decreased 40 per cent in fibroblasts from JODM when grown in the hydrocortisone medium. These studies indicate that skin fibroblast cultures from patients with diabetes exhibit abnormalities in cell proliferation. Furthermore, hydrocortisone appears to unmask diffeerences in protein synthesis that distinguish JODM and AODM fibroblasts in culture.


Use of dexamethasone in treatment of insulin lipoatrophy.

To treat insulin lipoatrophy, small amounts of dexamethasone (4 micrograms/unit) were added to insulin injections in nine patients. Six cases showed significant improvement of dexamethasone-treated sites over that of control sites in six months. With the continued use of dexamethasone-containing insulin, they remained mostly free of atrophic areas during the follow-up period of over two years. Our experience suggests the usefulness of this therapy.


Glucagon secretion induced by natural and artificial amino acids in the perfused rat pancreas.

The glucagon-secreting potency of 22 amino acids was investigated in the rat isolated perfused pancreas. Arginine and the structurally related amino acids were the most potent A2-cell stimulators that induced a biphasic and sustained glucagon release. Dose-response curver were different for L(+) and D(+)arginine, and the suppressor effect of glucose on the response to L(+) arginine was not detected in the presence of D(+) arginine or homoarginine. Citrulline was the only exception among the arginine-related amino acids; it displayed neither stimulatory nor inhibitory potency on glucagon release. The A2-cell response to D(+) amino acids and artificial analogues of arginine is a strong case for the theory of amino acid receptors' triggering the release of the hormone before (or in the absence of) further metabolism. The prominent rank of arginine and ornithine amont stimulatory amino acids and some other physiologic evidence suggest that A2-cell may play a regulatory role in the metabolsm of ammonia by the liver.


The effect of chronic oral antidiabetic therapy on insulin and glucagon responses to a meal.

Nineteen maturity-onset diabetic patients receiving oral hypoglycemic therapy in a university diabetes clinic completed a study to assess the efficacy of the oral agents and to determine their effects on pancreatic islet hormone secretion. All patients were receiving sulfonylureas, and seven were also receiving phenformin. The subjects were studied as outpatients in the clinic setting on four different occasions with collections of a baseline blood sample before a standard breakfast and a second sampling two hours postprandially, twice while on their prescribed medication and twice after having been withdrawn from the medication. The values obtained during the two studies on the two studies off medications were reproducible for each subject. Analysis of the results by paired differences revealed that mean 24-hour urine glucose values deteriorated significantly (p less than 0.005) after oral antidiabetic therapy was withdrawn; similarly, mean plasma glucose values, both at baseline and two hours postprandially, rose significantly (p less than 0.001) when subjects were off medication. Baseline serum insulin values were not changed, but postprandial levels were significantly higher on oral agents (p less than 0.005). Plasma immunoreactive glucagon was significantly lower both at baseline (p less than 0.02) and postprandially (p less than 0.005) when the subjects were on their antidiabetic medications. During the trial off medication, 16 patients became symptomatic, with three of these developing symptoms severe enough to require hospitalization. It is apparent from this study that oral hypoglycemic medications can play a role in controlling symptoms in maturity-onset diabetic patients and that the beneficial effect of these agents on hyperglycemia may, in part, be explained by their stimulation of endogenous insulin secretion and partial suppression of endogenous glucagon.


Gastric and duodenal ulcer healing under placebo treatment.

The healing time relation of 30 gastric and 15 duodenal ulcers was assessed by repeated endoscopic measurements of ulcer size on days 1, 21, and 42 of a 6-week period, during which a selected population of patients was being treated as outpatients on an inert placebo. Antacids in small amounts were only given in case of ulcer pain. There was evidence for a linear relationship between the absolute initial ulcer size and its reduction with the time in incompletely healed ulcers of both types. Their percentage reduction from the original size was 71% after 3 weeks in gastric and 83% in duodenal ulcers; 82% after 6 weeks in gastric and 83% in duodenal ulcers. The healing time course for both types of ulcers appeared to follow an exponential function. The calculated half-life was 1.7 weeks for gastric and 1.9 weeks for duodenal ulcers. The number of healed duodenal ulcers observed at the 3-week examination was significantly greater than that of healed gastric ulcers (P = 0.05). It is concluded that repeated measurements of the ulcer size before complete ulcer healing contribute to a optimal characterization of the healing time course. The calculated half-life of less than 2 weeks makes shorter control intervals reasonable for both types of ulcers. The ulcer half-life appears to be a more accurate criterion of the healing process than that of complete healing.


Effect of betazole on serum group I pepsinogen levels: relationship to gastric acid output in unoperated and postoperative patients.

A significant association has previously been found between a betazole-induced decrease in serum group I pepsinogen (PG I) levels and a low peak acid output (PAO) in symptomatic patients with vagotomy and gastric resection or a drainage procedure. This study compares the effect of betazole on serum PG I levels and gastric acid output in 245 unoperated patients (115 duodenal ulcer, 25 prepyloric ulcer, 32 gastric ulcer, 73 nonulcer) and in 73 symptomatic postoperative patients (15 subtotal gastric resection, 28 vagotomy and gastric resection, 30 vagotomy and drainage). A negative serum PGI response (2-hr serum PG I level less than 92% of basal) occurred in 10 (4.1% of the unoperated patients and in 31 (42.5%) of the postoperative patients. Seven (70%) of the former and 29 (93.5%) of the latter patients had a PAO of less than 10 mEq per hr, indicating that a negative serum PG I response is associated with a low PAO in both unoperated and postoperative patients. The PAO was greater than 10 mEq per hr in 93.1% of the 277 patients with a 2-hr serum PG I level of more than 92% of basal. Additional studies revealed that neither aspiration of gastric juice nor perfusion of the stomach with acid altered the serum PG I response. This suggests that topical acid does not modulate the effect of betazole on serum PG I levels. Finally, a negative serum PG I response has been shown to be paradoxical, in that gastric pepsin levels have been found to increase over basal concurrently with the decrease in serum PG I levels.


Effect of bisacodyl on the structure and function of rodent and human intestine.

The effect of bisacodyl on intestinal structure and function was investigated. Net water transport was measured under steady state conditions in vivo during single pass infusions of rodent and of human intestinal segments. Each segment served as its own control. Bisacodyl inhibited water absorption in rat jejunum, ileum, and colon. The degree of inhibition was linearly related to the logarithm of the bisacodyl concentration over the range of 0.05 to 2.0 mg per 100 ml. In human jejunal segments, bisacodyl, 1 mg per 100 ml, caused net water secretion. Bisacodyl, 5 mg every 6 hr, increased ileostomy output by 15% when it was fed to 5 patients with established ileostomies. By light microscopy, bisacodyl, 2 mg per 100 ml, erased cytoplasmic and nuclear detail within surface absorptive cells of rat intestine. By electron microscopy, the involved cells contained sparse and abnormal cytoplasmic organelles and nuclei which were deficient in chromatin. These results suggest that the laxative effect of bisacodyl is related to its ability to inhibit intestinal water absorption. Reduced absorption may be secondary to changes in surface absorptive cells.


Cell loss from normal and stressed gastric mucosae of the rat. An ultrastructural analysis.

The gastric mucosa is characterized by high rates of surface epithelial cell (SEC) loss. Exposure to acute stressors results in an increase in the incidence of SEC loss. However, little is known about the detachment of SEC from the epithelial sheet. We have used scanning and transmission electron microscopy to study mechanisms of SEC loss from the gastric mucosae of normal, fasted rats and of rats exposed to acute stressors. In fasted control animals SEC loss occurred primarily via extrusion of individual interfoveolar cells and only rarely by degeneration in situ. Each extruding cell was surrounded by a rosette-like configuration of adjacent, encroaching SEC. Mucosal integrity is maintained. Short term exposure to acute stressors results in an increase in the incidence of extrusive cell loss. Continued exposure to stressors also increases the incidence of degeneration in situ of isolated SEC or groups of SEC. Extrusion provides a mechanism for the necessary removal of gastric SEC. Acute stress may override or exhaust the extrusive mode, producing, via ischemia or surface diffusion of cytotoxic agents, SEC degeneration in situ and breaks in the mucosal barrier.


Pharmacological analysis of the effects of metoclopramide on the guinea pig isolated stomach.

A pharmacological analysis has been made of the effects of metoclopramide on the contractile activity of isolated longitudinal muscle strips prepared from the guinea pig stomach. Metoclopramide produced up to 3-fold increases in the amplitude of the spontaneous contractions exhibited by such a preparation. This phenomenon was antagonized by hyoscine but not by tetrodotoxin nor by hexamethonium. The contractile response to transmural stimulation was also enhanced by metoclopramide, as was the response to extrinsic acetylcholine. Neural inhibitory systems were not affected by metoclopramide. The apparent sensitization to extrinsic acetylcholine was, however, prevented by tetrodotoxin and hexamethonium, implying that this effect depends upon ganglionic stimulation by acetylcholine. These results suggest that metoclopramide may act by increasing the amount of acetylcholine released at the postgangionic cholinergic nerve ending.


Humoral regulation of vitamin B12 absorption by pregnant mouse small intestine.

In vivo experiments in which pregnant mice were fed an oral dose of 40 ng of [57Co]B12 demonstrated that there was a progressive increase in B12 absorption in the 3rd week of pregnancy. The absorptive mechanism resembled that in nonpregnant animals on the grounds that the mechanism was saturable, and presumably intrinsic factor (IF) dependent, and that it was localized to the ileum. In vitro studies of IF-57Co B12 binding to ileal homogenates confirmed the requirement for IF. Studies in late pregnancy showed that IF-[57Co]B12 binding to ileal homogenates, expressed as nanograms of IF-[57Co]B12 bound per milligram of protein, was twice that of nonpregnant animals. When ileal homogenates were prepared from mice on days 20 to 21 of gestation, IF-[57Co]B12 binding fell to nonpregnant levels within 2 to 4 hr of hysterectomy. Hourly injections of serum from mice in late pregnancy, or from a female at the 37th week of gestation, maintained IF-B12 binding at levels reached during pregnancy, whereas nonpregnant serum was ineffective.


Compensatory postresectional hyperplasia and starvation atrophy in small bowel: dissociation from endogenous gastrin levels.

Because exogenous gastrin has been implicated as a tropic hormone for mucosal cells of the intestinal tract and as a regulator of mucosal growth, we studied the effects on the gut of endogenous 23-fold variations of serum gastrin produced by various gastric operations. These were pyloroplasty, vagotomy and pyloroplasty, antrectomy, and fundectomy. After jejunal resection, RNA content, DNA content, incorporation of [3H]thymidine into DNA, crypt depth, and villus height in midgut and ileum were independent of gastrin levels. Loss of RNA and DNA during starvation was not prevented by high endogenous concentrations of serum gastrin, and DNA specific activity was not affected. Tropic effects distal to the duodenum produced by exogenous gastrin and pentagastrin may be results of supranormal levels.


Influence of experimental bypass on cellular kinetics and maturation of small intestinal epithelium in the rat.

The effect of experimental bypass on the intestinal epithelium of the rat was investigated from 2 to 360 days after creating a Thiry-Vella fistula. Within 7 to 14 days the number of cells per villus column and, to a lesser degree, the number of cells per crypt column decreased and subsequently these values remained constant up to 1 year. Radioautography did not show any changes in the percentage of labelled cells per crypt column, after incorporation of [3H]thymidine. In addition, the relative size of the proliferative cell compartment in the crypt, the number of crypts per unit length of small intestine and the crypt-villus ratio remained unchanged. Scintillation counting of isolated crypts from the Thiry-Vella fistulas showed that the number of cells per crypt was markedly reduced. Scanning electron microscopy revealed that the three-dimensional size of the villi was also reduced. The activities of nonspecific esterases and neutral alpha-glucosidase remained unchanged in crypt cells and were above control values in villus cells. The development of these biochemical characteristics of the villus cells seems to be independent of the luminal contents. It was concluded that deprivation of food passage and gastric, pancreatic, and liver secretions from a small intestinal segment primarily causes a reduction in the number of cells per crypt, which leads to a lower cell production. In turn, this is responsible for the reduction of the functional villus cell compartment.


Prednisone for chronic active liver disease: pharmacokinetics, including conversion to prednisolone.

To determine the effect of impaired liver function on conversion of prednisone to prednisolone, and to investigate the relationship of this to responses to treatment with prednisone, we measured serum prednisone and prednisolone by radioimmunoassay after 10 mg of prednisone was given by vein to 10 healthy volunteers, 6 untreated patients with severe chronic active liver disease (CALD), 10 patients with prednisone-induced remission of CALD, and 3 patients with CALD deteriorating despite treatment with prednisone. Prednisone disappearance was comparable in all groups and substantial values for serum prednisolone appeared in all groups within 0.3 hr. Minor differences between the groups included lower than normal serum prednisolone in severe CALD and treatment failure; a higher percentage of nonprotein bound prednisolone in such patients; and an association between earlier treatment with prednisone and an increased disappearance rate of prednisolone. We conclude that no major defect of prednisone metabolism occurs in CALD and that failure of this condition to respond to therapy with prednisone is caused by other factors.


Intracellular electrolyte abnormalities in fulminant hepatic failure.

The sodium, potassium, and water content of peripheral blood leukocytes was determined in 30 patients with fulminant hepatic failure. Although values for potassium were reduced, statistically significant increases were found in sodium and water content. Serial studies showed that, with recovery of liver function, the leukocyte sodium content fell initially to below normal, with values subsequently returning to the normal range some weeks later. Leukocyte sodium content was inversely correlated to the plasma sodium concentration, suggesting that a shift of sodium into the intracellular compartment might contribute toward the hyponatremia that was found in many patients.


Induction of hepatic microsomal enzymes after brief administration of rifampicin in man.

The inducing effect of rifampicin (600 mg per day) on the hepatic microsomal drug-metabolizing system has been studied in 7 patients with normal liver after 6 days of oral administration. Using the plasma disappearance rate of antipyrine (15 mg per kg of body weight) as an index of liver microsomal metabolism, a significant decrease in the half-life of antipyrine has been observed: 11.7 +/- (1 sd) 4.7 hr before treatment as compared to 6.9 +/- 2.3 hr on the 7th day. Concomitantly, the half-life of rifampicin was 5.3 +/- 2.1 and 2.7 +/- 0.8 hr on the 1st and 7th day, respectively. These results demonstrate that rifampicin administration for only 6 days leads to the induction of the microsomal mixed function oxidase system in liver. These data are in agreement with the recent hypothesis that rifampicin may play a role in the hepatotoxicity of isoniazid by enhancing its microsomal transformation to a toxic metabolite.


Dietary cholesterol affects chenodeoxycholic acid action on biliary lipids.

Chenodeoxycholic acid (CDC) decreases biliary saturation and dissolves gallstones in one-half of the treated patients. Dietary cholesterol also affects biliary lipids and is a possible factor explaining unsuccessful CDC therapy. The aim of this investigation was to study the effect of high and low dietary cholesterol on the CDC-induced decrease of biliary saturation and activity of hepatic hydroxymethylglutaryl coenzyme A reductase (HMG-CoAR). Seventy two hamsters in six groups were fed for 1 month one of three diets: 0.8 mg of cholesterol per g of food, 2.4 mg of cholesterol per g, or cholesterol-free. On each diet hamsters received no CDC or CDC 30 mg per kg per day. When animals were killed, biliary lipids were determined and the activity of hepatic HMG-CoAR was assayed. CDC administration decreased the saturation index (SI)(P less than 0.01) in hamsters on the high cholesterol and standard diets but not on the cholesterol-free diet. The SI in CDC-treated hamsters on the high cholesterol (0.78 +/- 0.03) and cholesterol-free (0.68 +/- 0.02) diets were greater (P less than 0.02) than in CDC-treated hamsters on the standard diet (0.48 +/- 0.03). CDC decreased (P less than 0.01) HMG-CoA reductase activity on each diet. In comparison to HMG-CoAR activity (190 +/- 7.6 pmoles per mg per min) in CDC-treated hamsters on the standard diet, the activity in CDC-treated hamsters on the high cholesterol diet (176 +/- 5.8 pmoles per mg per min) was decreased ( less than 0.05), whereas the activity on the cholesterol-free diet (495 +/- 11.5 pmoles per mg per min) was greater (P less than 0.01). It is concluded that: (1) dietary cholesterol is necessary for optimum CDC inhibition of HMG-CoAR; (2) high cholesterol and cholesterol-free diets prevent maximum CDC decrease of the biliary saturation index; (3) dietary cholesterol alterations may therefore be one cause of the failure of CDC dissolution of gallstones.


Portal hypertension and esophageal varices in hemangiomatosis of the spleen.

Bleeding esophageal varices and severe portal hypertension developed in a patient with capillary hemangiomatosis of the spleen. No signs of cirrhosis were found at postmortem examination, and preoperative celiac angiography suggested an intrasplenic shunt as the cause of the high portal pressure. Although a very rare condition, this possibility should be considered in the differential diagnosis of esophageal varices.


Usefulness of rectal biopsy in inflammatory bowel disease.

With careful "standardized" technique and proper handling, rectal biopsies can be useful diagnostic and therapeutic adjuncts in the initial evaluation and then long term observation of patients with chronic inflammatory bowel disease.


The development in very young children of tacit knowledge concerning visual perception.

The purpose of this study of early social-cognitive development was to assess the very young child's behaviorally expressed knowledge of people's visual-attentional acts and abilities. Boys and girls (N = 60) 1, 1 1/2, 2, 2 1/2, and 3 years of age were tested in their homes with their mother's help. Three sorts of tasks were used: 1. Percept production. The child's task was to produce a visual percept in the other. Examples include pointing to objects ("productive pointing") and a wide variety of object-showing problems. 2. Percept deprivation. The opposite, exemplified by a variety of object-hiding problems. 3. Percept diagnosis. The child's task was to determine what the other was already visually attending to, either by looking where his or her finger was pointed ("receptive pointing") or where his eyes were directed. It was found that the majority of 1-year-olds produced and comprehended pointing, and would sometimes hold out a toy to show it, but did little else. The 3-year-olds were at ceiling on virtually all tasks. At 1 1/2 years, children usually showed a picture by holding it flat so that both they and the other could see it. Jrom 2 on, they usually turned it toward the other in the adult fashion. Very few children of any age showed egocentrically--i.e., orienting the picture so only they could see it. By age 2, the children solved what were presumably novel showing problems for them: e.g., successfully showing to another a picture pasted on the inside bottom of a hollow cube. Hiding ability emerged later than showing ability but seemed well established by age 3. The role of the other's eyes in seeing appeared to be quite well understood at least by age 2-2 1/2. As examples, children of this age took the other's hands away from her or his eyes before trying to show her something, and could usually tell where she was looking from her eye orientation alone. These age trends presumably reflect important developments in the area of social interaction and communication, as well as with respect to cognition about percepts.


Psychological changes accompanying pregnancy and motherhood.

This study was an attempt to investigate some of the psychological changes that occur during first pregnancy and the early postpartum months. The major theoretical assumption was that pregnancy and early motherhood may be viewed as a series of developmental tasks, and that the way in which these are coped with will be predictive of adaptation to the maternal role. The aims of the study were (a) to identify the affective changes that occur in women during first pregnancy and to assess the extent to which pregnancy and motherhood are experienced as a period of psychological crisis; (b) to trace the development of maternal feeling; and (c) to assess the extent to which characteristics measured early in pregnancy are predictive of attitudes and adjustment to later stages of pregnancy and the degree to which these factors are themselves predictive of adaptation to parenthood. The sample consisted of 19 white, middle class primigravidas, with an age range of 22-33 years. Only women who had no previous gynecological or psychiatric difficulties and who were currently living with their husbands were accepted as Ss. Each woman was interviewed at each trimester of pregnancy, on the third postpartum day, and at 2 months postpartum. A follow up questionnaire was mailed at 7 months postpartum, The data obtained were derived from ratings of extensive interview schedules and a number of personality measures. Results indicated that although emotional upheaval and rapid change were characteristic of pregnancy, for some women a growing sense of adulthood, of fulfillment, and integration of a new maturational stage clearly co-existed with the emotional disequilibrium. Analysis of the interrelationship among characteristics shown in early pregnancy, adjustment to the pregnancy overall, and adaptation to parenthood indicated that the degree of personality integration achieved by early pregnancy was predictive of the extent to which psychological growth was experienced throughout pregnancy and early parenthood.


[Principles of the therapy of osteoporosis. 1. Definition, classification, diagnosis].

More and more the treatment of osteoporotic changes of the skeleton becomes a topic of discussion of an emotional nature. Whatever reasons may exist, it can be taken for granted that a considerable interest can be found in the pertinent problems coming up, and that a difference of opinion exist on some issues. However, certain principles should not be disputed, since nowadays there is hardly any doubt as to their validity. To represent these and to compare them with the methods of treatment of an osteoporosis discussed today is the purpose of this information. This first part contains definition, classification and diagnosis.


[Mondor's disease].

Mondor's disease is described on the basis of 4 patients of our hospital. The typical finding is a subcuttaneous cord of about 5 min in diameter along the chest wall. The lenght varies from 10 to 40 cm. There is no significant preference of one sex. Often there are no symptoms like pain or tension of the skin. The clinical and histological examinations show in most cases a thrombosed vein at the junction of the subcutaneous tissue. Treatment does not result in a significant change of the natural course. Our study confirms, that Mondor's disease is benign and does not connote systemic disease or cancer.


[Epididymitis and fertility. Treatment results in acute unspecific epididymitis].

Spermiograms of 46 patients were examined 8 days, 4 weeks, 3 months, 6 months and 1 year after acute unilateral non-specific epididymitis. The treatment consisted in 24 cases besides antibiotic treatment with doxycycline in local infiltrations of the spermatic cord with 1% Scandicain solution. Primarily there was found an inhibiting effect on spermatogenesis. In more than 2/3 of the cases a varying oligoasthenospermia was shown 8 days after beginning of the infection. In the further course this disturbance of fertility improves; if local infiltration of the spermatic cord with 1% Scandicain solution is performed, the improvement is more evident. Still, there remain about 20% of the cases in which the fertility disturbance is so severe, that sterility results. One year after the beginning of the infection spermagglutinating antibodies could be shown only in 1 case. The seminal fluid fructose was controlled over 1 year following the beginning of infection and found to be normal in all cases.


[Pathogenetic concept of experimental mycoplasma arthritis].

Biochemical studies of rats with mycoplasma arthritis revealed new findings in pathogenesis and pathophysiology. Mycoplasma infection which resembles rheumatoid arthritis, leads to an inhibition of the DNA-repair. We were able to proof this enzyme--kinetically and autoradiographically. We also observed for the first time the occurrence of DNA-antibodies in this type of arthritis. It is possible that there is a relation between inhibition of DNA-repair and the occurrence of DNA-antibodies. Thus mycoplasma infection seems to influence DNA-metabolism. There are interesting parallels concerning DNA-antibodies and DNA-repair between experimental mycoplasma arthritis and human systemic lupus erythematosus and rheumatoid arthritis.


[Care of the hemodialysis patients by the family physician].

Cooperation between the family physician and the kidney-center begins with the recognition of a renal disease and pre-dialysis treatment. Our patients usually are sent for the preparation of an arterio-venous shunt operation (Cimino or modifications) when serum creatinin levels amount to 8 to 10 mg/100 ml. Peripheral veins on both forearms should be reserved for these procedures early in the course of renal disease and vascular punctures should be avoided. Dialysis treatment is performed either at the kidney-center, at one of our partner-centers, at the central self-care facility operating under the care and supervision of the kidney-center or as home-dialysis-treatment. Each patients continues to receive technical and medical services of the center. Central self-care dialysis as well as home-dialysis are organized by the Kuratorium for Heimdialyse e.V. in this area. This organization also provides an on-cell-service of technicians. Nurses and physicians take regular rotations to staff the dialysis- and the self-care-units. The family physician takes care of the hemodialysis patinet in cooperation with the hospital. In case of medical problems the patient is transferred to the kidney-center. The patient must be well instructued on problems and complications which might occur during hemodialysis, either due to the basic disease or in connexion with hemodialysis. In some cases of complications patients must be admitted to the center without delay. Emergency situations usually can be avoided as technical standard of dialysis equipment and standard of training of patients or their parners is high. Medications, such as phosphate binders (aluminium hydroxide), iron vitamins and allopurinol are provided if necessary. Patients are advised to limit intake of fluids and potassium containing foods. The sodium intake depends on blood pressure-values. In case of hypertension there will be salt restriction, in case of hypotension the salt intake is increased. Chronic intermittent hemodialysis treatment can result in successful rehabilitation. Further improvement concerning personal and medical problems can only be expected from kidney transplantation.


[Hemolytic components of anemia in patients with long-term hemodialysis].

Hemolysis was investigated in 12 patients on chronic dialysis. A hemolytic component was evaluated by estimation of free hemoglobin concentration, elevated activity of red cell enzymes as well as by reduction of red cell life span to 2/3 of normal. Only in one case where the patient suffered also from familial hemolytic jaundice, splenectomy was necessary.


[Day-care mother--a successful model of child rearing?].

The scientific reports published so far on the project "day care mothering" do not allow to draw scientific conclusions, whether this educational model can achieve nearly equal results as family education. The author critisises, that such a clame is made. It seems necessary, to pay more importance to the function of the mother for her children, to give the children a sound emotional basis for their future development.


[Nutrition of premature infants with human milk and Milupa Meb].

In 45 prematures and small for date-infants weighing more than 1750 g a special formula (Meb) was compared with pooled human milk. Weight gain during feeding human milk occurred more slow that during feeding special formula. This may be due to the different protein- and mineral-intake, while the amounts of calories were similar. Differences of blood-glucose, total bilirubin, beta-cholesterol and of one protein-fraction (alpha-2-globulin) could be statistically as certained. No difference resulted in hemoglobin, red blood cell count, hematocrit, total cholesterol, triglycerides, phospholipids, beta-lipoproteins, transferrin, and protein-electrophoresis. All parameters together suggest, that this special formula for low-birth-weight infants can be used in nutrition of these newborns.


[Diagnostic and therapeutic problems of the stress ulcer].

Several examples show the five pathogenetic patterns of stress-ulcer induction. Own clinical experiences with 60 cases of stress-ulcers from 1973 to 1974 are described. Our studies on the effect of Vit. A und STH on stress-ulcera are compared with those of other authors and are critically discussed. The different excretion patterns of the group I and group II pepsinogens are pointed out. Difficulties exist not only from here but also from the unknown correlation between serum and urine pepsinogens as far as the attempt is concerned, to work out a pattern to diagnose stress-ulcera before they become clinically manifest.


[Cardiac survival time under the influence of carbochromen: Studies and results].

In rabbits carbocromen was tested to influence heart tolerance to hypoxic conditions. The time between onset of asphyxia (induced by succinylcholin) and ECG silence was recorded. Carbocromen increased this time period by about 60 p c(p less than 0.001). It is discussed whether this effect is due to newly discovered metabolic changes or to the well known coronary vasodilation, both experimentally demonstrated for carbocromen.


Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs.

Pre-culture of spleen cells for 24 h before addition of activator greatly improved the mitogenic response to phytohaemagglutinin (PHA) and sheep erythrocytes (SRBC). Spleen cells were separated into plastic-adherent and non-adherent populations and in some experiments purified further to 'macrophages' and 'lymphocytes' respectively. A critical mixture of lymphocytes and macrophages (10:1) gave twice the mitogenic response of lymphocytes alone to PHA and SRBC in cultures of foetal, juvenile and adult guinea-pig spleen cells. Vigorous mitogenic responses to PHA and SRBC were found at 36 days of gestation. The mitogenic response increased from 46-56 days of gestation to above adult level. Lipopolysaccharide (LPS) obtained by the Westphal method was a superior mitogen to LPS extracted by the Boivin technique. Plaque-forming cells (PFC) could not be induced in vitro at any age even with the addition of LPS. Evidence was obtained for mitogenic and blastogenic lymphokines produced by lymphocytes activated with PHA or produced by lymphocytes activated with PHA or SRBC (but not LPS). These lymphokines were produced by activated splenic lymphocytes of a 40-day-old foetus; older animals showed no evidence for a quantitative increase in lymphokine production. The onset and maturation of mitogenic responsiveness in the guinea-pig and human foetus is compared by age-equivalence.


Biphasic pattern of activation of the reticuloendothelial system by anaerobic coryneforms in mice.

Macrophage activation as measured by increased rates of clearance of carbon was explored in five inbred and two outbred strains of mice pretreated with anaerobic coryneform bacilli. Constant differences were found according to strain from DBA (lowest response) to Sha-Sha (highest response). The investigation was continued with mice of CBA strain which also provided highly reproducible and high-level responses. In this strain activation occurred in two phases: an early activation which reached maximum levels at 2 days and attributable to a lipid component of the bacteria, and a late phase reaching maximum at 14 days which appeared to coincide with greatly increased weight of the liver and spleen, due to infiltration by mononuclear cells. Evidence is provided that the early phase of macrophage activation is due to a lipid extract from the anaerobic coryneforms with chemotactic activity.


Lymphocyte surface membrane changes in dividing cells and following regidification with mitogens.

Iodination of surface membrane protein has allowed the dynamics of lymphocyte surface membrane protein to be examined. A comparison has been made between the rate of membrane turnover in fresh peripheral blood lymphocytes and lymphocytes in other states. When receptor redistribution is inhibited with high concentrations of mitogens, 'rigidification' of the membrane occurs and protein turnover is very much reduced. Loss of surface membrane protein is also much slower in lymphocytes which have been stimulated with mitogens and are undergoing active DNA synthesis. It is thought that these observations may relate to immunological inactivity and may have implications for pathological unresponsiveness.


The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG;.

IgM rheumatoid factors (RF) were isolated from the sera of patients with rheumatoid arthritis and a serologically active Fabmicron RF fragment prepared by papain digestion. A radioimmunoassay was developed for the determination of interaction of 19S IgM RF and Fabmicron RF with human 7S IgG, heat-aggregated IgG, rabbit 7S IgG, and human pFc'. RF isolated under neutral conditions had a very low binding constant for human 7S IgG (of the order of 10(2) to 10(3) 1 mole-1) and a considerably higher value (ca. 10(5)) for the aggregated protein and monomeric rabbit IgG. RF obtained under acid conditions which dissociate the complexes with endogenous Ig, had a higher avidity for human IgG monomer as expected and also a comparable reactivity with rabbit IgG. Monovalent Fabmicron fragments of 'acid' RF had closely similar affinities for 7S and aggregated IgG suggesting that the enhanced binding with the aggregated protein is essentially dependent on its multivalency rather than the exposure of a new determinant lacking in the native molecule.


Metabolism of exogenous single stranded DNA in normal and NZB/W mice.

Metabolism of ss-DNA in Swiss Webster mice and NZB/W mice was studied. The delay in intra-hepatic catabolism of ssDNA in old NZB/W female mice seems to correlate with the presence of increased anti-DNA antibodies; similar to effects produced in Swiss Webster mice that have been pretreated with a small dose of carbon black. Neither impairment in the phagocytic ability nor a decrease in DNase activity are found in the mice who are relatively unable to metabolize ssDNA. These observations are discussed in relation to possible pathogenesis in NZB/W mice.


Ferritin and ovalbumin binding blood lymphoid cells in fetal sheep.

Lymphoid cells from blood of fetal sheep between 70 and 140 days of gestational age were examined for surface immunoglobulin (sIg) and for surface binding of horse spleen ferritin FITC and ovalbumin FITC. Fetuses of all ages had cells which bound ferritin or ovalbumin, the proportion of these cells in the lymphocyte preparations from blood decreasing with increasing age of the fetus. A similar decline in the proportion of sIg cells with age was found. The simultaneous occurrence of fetal lymphoid cells binding ferritin or ovalbumin is discussed in relation to the sIg population and to the property of the sheep fetus to respond sequentially to different antigens by the production of antibody.


A retrospective study of probable glucocorticoid-induced hepatopathy in dogs.

Hepatopathy associated with glucocorticoid therapy or with naturally occurring hyperadrenocorticism was detected retrospectively in hepatic biopsy specimens from 22 of 60 dogs, by histologic review. The hepatopathy was characterized by centrilobular vacuolization, perivacuolar glycogen accumulation within hepatocytes, and focal centrilobular necrosis. The predominant clinical findings were hepatomegaly, increased serum enzyme activity associated with hepatic disease, and increased bromsulphalein retention. The hepatopathy appeared to be reversible.


Ophthalmic nodular fasciitis in the dog.

Ophthalmic nodular fasciitis in 5 dogs was often manifested as subconjunctival masses of variable growth rate. Each mass was raised, pink, and hyperemic. Five masses involved the bulbar conjunctiva, and 2 masses involved the eyelids. Local excision of each mass successfully resolved the condition.


Metabolic products of microoorganisms. 163. Desferritriacetylfusigen, an antibiotic from Aspergillus deflectus.

Aspergillus deflectus CBS 109.55 when grown on an iron-free medium produces several antibiotics; one of these was isolated and identified as desferritriacetylfusigen. It inhibits the growth of bacteria, whereas yeasts and fungi are not or only weakly affected.


Nucleosides. CVI. Syntheses of 1-N-methyl-5-(beta-D-ribofuranosyl)uracil (1-N-methyl-phi-uridine) and its identity with a metabolite elaborated by Streptomyces platensis var. clarensis.

Very recently, 1-N-methyl-psi-uridine was isolated from the culture filtrate of Streptomyces platensis var. clarensis along with an antibacterial and antiviral antibiotic, U-44590. We achieved chemical syntheses of 1-N-methyl-psi-uridine by selective methylation of psi-uridine in two different routes and established the identity of the synthetic nucleoside with the natural product.


Polyene antibiotics. VIII. The structure of rimocidin.

A structure is assigned to the tetraene antibiotic rimocidin, based in part on 13C NMR spectroscopy and field desorption mass spectrometry. The structure assigned revises a structure proposed in 1966 which was anomalous in relation to those of other polyene antibiotics.


Metabolism of putrescine in the central nervous system.

The metabolism of putrescine in rat brain was studied systematically by the intraventricular injection of radioactive diamine. Putrescine injected into the brain was metabolized mainly to polyamines, spermidine, and spermine. A small portion of the radioactivity of putrescine was incorporated into gamma-glutamylputrescine, putreanine, gamma-aminobutyric acid, and homocarnosine. Comparison of the specific radioactivities of gamma-aminobutyric acid and homocarnosine after the injection of radioactive putrescine with those after the injection of radioactive glutamic acid indicated that there may be a metabolic pool of gamma-aminobutyric acid (putrescine-gamma-aminobutyric acid system) which is different from the glutamic acid-gamma-aminobutyric acid system and which is effectively used for synthesis of the dipeptide.


Effect of cycloheximide on the synthesis and processing of 5S RNA in HeLa cells.

The synthesis and incorporation into ribosomes of 5S RNA were investigated under conditions of protein synthesis inhibition by cycloheximide. Within 1 h after addition of cycloheximide to the culture medium, the synthesis of 5S RNA in HeLa cells, as measured by the pulse labeling of total 5S RNA, was inhibited by 25-30 per cent, but the degree of inhibition did not further increase for several hours. The synthesis of 4S RNA was less markedly inhibited, especially at earlier time periods. Although the appearance of labeled 5S RNA in cytoplasmic ribosomoes after chase was inhibited by approximately 60% or more this presumably reflects the inhibition of 60S ribosomal subunit formation by cycloheximide. Some utilization of preformed 5S RNA for new ribosomes was suggested after resumption of protein synthesis. In confirmation of the results of Kumar and Wu (1), a low dose of actinomycin D inhibited the appearance of 28S RNA in the cytoplasm rather specifically, whereas cycloheximide showed an apparently opposite differential effect.


Location of the iron-sulfur cluster in Spirulina platensis ferredoxin by x-ray analysis.

A chlorplast-type ferredoxin containing two non-heme iron and two labile sulfur atoms per molecule was prepared from Spirulina platensis. The protein crystallized in the orthorhombic system with cell dimension a=62.32, b=28.51, and c=108.09A. The space group is C222, and one asymmetric unit contains one molecule. The electron density maps at 5A and 3.5A resolutions were synthesized utilizing the best phase angles calculated by the single isomorphous method coupled with the anomalous dispersion method. The difference Fourier synthesis with the anomalous scattering difference of the native data showed the location of the iron atoms clearly. Comparing the location of the iron atoms with the best phase angle electron density map, it was concluded that the active center of the present molecule is close to the surface of the molecule.


Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in dimyristoyllecithin vesicles and the determination of "microviscosity".

The nanosecond time dependence of the fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dimyristoyllecithin vesicles was determined at temperatures above and below the midpoint of the gel-liquid crystalline transition. In neither case could the decay of the total fluorescent emission or the decay of the emission anisotropy be described adequately in terms of single exponential decay laws. At the lower temperature, the emission anisotropy did not approach zero in the time window available for measurement, a finding which may indicate that the range over which rotation of the probe can freely occur is restricted. The results are discussed in relation to the concept of microviscosity of bilayer membranes.


Active transport of 5-hydroxytryptamine by plasma membrane vesicles isolated from human blood platelets.

Plasma membrane vesicles isolated from human platelets accumulate 5-hydroxytryptamine when an electrical potential (interior negative) or an Na+ gradient (out greater than in) is imposed across the vesicle membrane. Kinetic studies reveal a Km of 0.5 micronM for the transport process. Uptake is inhibited strongly by tricyclic antidepressants and by ionophores such as gramicidin which catalyze transmembrane exchange of Na+ for K+. Transport is absolutely dependent upon external Na+ and Cl- and is only mildly, if at all, inhibited by reserpine, cinanserin, ouabain, or arsenate. Experiments are presented which suggest that a single positive charge crosses the vesicle membrane with each molecule of 5-hydroxytryptamine. The results provide direct evidence for Na+-coupled active 5-hydroxytryptamine transport by the platelet plasma membrane.


Preparative polyacrylamide gel electrophoretic purification of human alpha- and beta-globin messenger RNAs.

Previous studies have reported the use of globin chain-specific complementary DNAs to quantitate the amount of human globin mRNA and human globin genes in normal and abnormal cells. In order to obtain larger amounts and more purified globin mRNAs as templates for these experiments, preparative polyacrylamide gel electrophoresis in formamide has been used to separate alpha- and beta-globin mRNA from polyadenylate containing RNA of human reticulocytes. Fifty to one hundred-fifty micrograms of RNA can be applied to the preparative gel and the recovery of the globin mRNA is about 50%. The isolated alpha- and beta-globin mRNAs were assayed in a wheat germ cell-free system, and the alpha- and beta-globin synthesized quantitated by cellulose acetate electrophoresis. The purified alpha- and beta-globin mRNAs direct globin synthesis which is more than 90% either alpha- or beta-globin, respectively. The cDNAs prepared using each of the isolated mRNAs as template are also shown to be specific for alpha- or beta-mRNA sequences. The gel electrophoresis technique used permits the relatively large scale isolation of alpha- or beta-globin mRNAs from human cells.


5-O-beta-D-Galactofuranosyl-containing exocellular glycopeptide of Penicillium charlesii. Incorporation of mannose from GPD-D-mannose into glycopeptide.

Three-day-old Penicillium charlesii mycelia were broken with Al2O3 in a buffered system and the membranes were separated on a linear gradient of sucrose concentrations. The most active guanosine 5'-(alpha-D-mannopyranosyl pyrophosphate):glycopeptide mannopyranosyltransferase (GPD-D-mannose mannosyltransferase) was found in two unresolved membrane fractions ( p congruent to 1.1 g/cm3). This preparation incorporated [14C]mannose from GDP-D-[14C]mannose both into endogenous acceptors and added peptidophosphogalactomannan. Mannosyltransferase activity is optimum at pH 7.0 in 0.05 M Tris/maleate buffer, and 17 mM Mn2+. Replacement of Mn2+ with Fe2+, Mg2, Co2+, Ca2+ or Ni2+ greatly reduced the mannosyltransferase activity. [14C]Mannose incorporation from GDP-D-[14C]mannose into acceptors is linearly dependent on enzyme concentration. Mannosyl incorporation into peptidophosphogalactomannan is linear for 3 h and continues for at least an additional 4 h. In contrast, the rate of mannosyl incorporation into endogenous acceptor(s) decreases after 60 min and there is no incorporation after 2 h. A series of possible acceptors related to peptidophosphogalactomannan were tested and it was found that treatment of peptidophosphogalactomannan with 0.01 N HCl at 100 degrees did not appreciably decrease the effectiveness of the acceptor even though this treatment removes the galactofuranosyl residues. In contrast, treatment of peptidophosphogalactomannan with 0.5 N NaOH rendered the products nearly incapable of accepting mannosyl residues from GDP-D-mannose. Derivation of peptidophosphogalactomannan with 2,4-dinitrobenzene also decreased its effectiveness as a mannosyl acceptor. [14C]Mannose from GDP-D-[14C]mannose was incorporated into both the oligosaccharide and phosphogalactomannan regions of peptidophosphogalactomannan. Treatment of the [14C]peptidophosphogalactomannan product, with 0.4 N NaOH released [14C]mannosyl-containing residues which eluted in the mannobiose and polysaccharide fractions from BIo-Gel P2. Approximately 90% of the 14C was in mannobiose. The [14C]mannose was shown to be transferred to the mannosyl-(seryl/threonyl) region of the acceptor. Acetolysis of [14C]peptidophosphogalactomannan resulted in the isolation of [14C]mannose, [14C]mannobiose, and [14C]polysaccharide. Small quantities of 14C were obtained in mannotriose and mannotetraose. The time course of [14C]mannose incorporation into the oligosaccharide region of peptidophosphogalactomannan showed a continual increase over a 4-h interval. In contrast, there was no major increase after 1 h in [14C]mannose incorporated into the polysaccharide region. The enzyme catalyzing incorporation of mannose into the polysaccharide region of peptidophosphogalactomannan was solubilized by treatment of the membranes with Triton X-100.


Glucosyltransferase activity in calf pancreas microsomes. Formation of dolichyl D[14C]glucosyl phosphate and 14C-labeled lipid-linked oligosaccharides from UDP-D-[14C]glucose.

Calf pancreas microsomes incorporated radioactively labeled D-glucose from UDP-D-glucose into products extracted with chloroform/methanol (2:1, v/v), chloroform/methanol/water (10:102.5, v/v), and into the residual precipitate, with a pH optimum in Tris/maleate buffer of about 5.3. The chloroform/methanol extract contained a single 14C-labeled acidic product, which was identified as dolichyl beta-D-glucosyl phosphate. It was stable to mild alkali, yielded D-[14C]glucose upon mild acid hydrolysis, and a 14C-labeled compound with the chromatographic mobility of 1,6-anhydro-beta-D-glucopyranosyl upon hot alkali treatment. The [14C]glucolipid had the same chromatographic mobility as dolichyl beta-D-[14C]mannosyl phosphate, and its formation was stimulated by exogenous dolichyl phosphate. The chloroform/methanol/water extract contained radioactive lipid-bound oligosaccharides which were retained on DEAE-cellulose more strongly than dolichyl D-[14C]glucosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a D-glucose-labeled oligosaccharide fraction which was shown by gel filtration to be slightly larger than most of the D-mannose-labeled oligosaccharides. About 80% of the radioactive D-glucose residues could be removed with alpha-glucosidase, but not with beta-glucosidase. Pancreatic dolichyl beta-D-[14C]glucosyl phosphate incubated with calf pancreas microsomes served as direct donor of D-glucosyl residues to lipid-bound oligosaccharides and to the precipitate. These oligosaccharides had the same size as those labeled from UDP-D-[14C]glucose, and the D-[14C]glucose residues could also be removed with alpha-glucosidase.


Fluorescent substrate for nascent peptidoglycan synthesis. Uridine diphosphate-N-acetylmuramyl-(Nepsilon-5-dimethylaminonaphthalene-1-sulfonyl)pentapeptide.

The synthesis of UDP-MurNAc-Ala-DGlu-Lys[Nepsilon-dimethylaminonaphthalene sulfonyl (Dns)]-DAla-DAla provides a method for the specific introduction of a fluorescent reporter group into the membrane environment of nascent peptidoglycan synthesis. To assess the degree of perturbation of this environment caused by the introduction of the dansyl substituent, this nucleotide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase (UDP-MurNAc-Ala-gammaDGlu-Lys-DAla-DAla:undecaprenyl phosphate phospho-MurNA-C-pentapeptide transferase) and in the membrane-associated synthesis of nascent peptidoglycan. Phospho-MurNAc-pentapeptide translocase in membrane fragments from Staphylococcus aureus Copenhagen catalyzed the transfer of phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla to undecaprenyl phosphate with a Vmax/Km of 3.8 and a Vmax of 1.6 times the values for UDP-MurNAc-pentapeptide. In the exchange reaction catalyzed by the translocase, the Rmax/Km and Rmax for the dansylated substrate were 1.8 and 0.78 times the respective values for the reference nucleotide. The equilibrium constant for the transfer reaction utilizing UDP-MurNAc-(Nepsilon-Dns)pentapeptide was 5.9 +/- 0.13 compared to 1.1 +/- 0.02 for UDP-MurNAc-pentapeptide. With respect to the proposed reaction model (Pless, D. D., and Neuhaus, F. C. (1973) J. Biol. Chem. 248, 1568-1576), the increase in Keq is consistent with a decrease in the affinity of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla for the translocase. The fluorescence emission maximum of the phospho-MurNAc-(Nepsilon-Dns)pentapeptide moiety of UDP-MurNAc-(Nepsilon-Dns)pentapeptide was blue-shifted from 525 to 495 nm upon transfer from UMP to undecaprenyl phosphate with a 6-fold increase in quantum yield. These spectral changes provided a sensitive and continuous assay for the formation of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dans)-DAla-DAla. The nascent peptidoglycan synthesizing system from Gaffkya homari utilized the dansylated nucleotide with a Vmax/Km of 0.05 and a Vmax of 0.10 times the values for UDP-MurNAc-pentapeptide. These results demonstrate that phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla linked to the undecaprenyl phosphate will serve as a precursor for the synthesis of nascent peptidoglycan and that the dansyl moiety will report on the membrane environment it experiences during this synthesis.


Oxygen equilibrium curve of normal human blood and its evaluation by Adair's equation.

Oxygen equilibrium curves of fresh, normal human blood have been measured by new methods which allow the control of pH, pCO2, and 2,3-diphosphoglycerate and which yield higher accuracy at the extremes of saturation than was possible previously. The curve determined by these techniques lies slightly to the right of the standard curve of Roughton et al. (Roughton, F.J.W., Deland, E.C., Kernohan, J.C., and Severinghaus, J.W. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Astrup, P., and Rørth, M., eds) pp. 73-83, Academic Press, New York). The greatest difference is at low oxygen saturation, probably owing to the fact that the latter data were obtained under conditions which would lead to depletion of cellular 2,3-diphosphoglycerate. The range of p50 (oxygen pressure at half-saturation) values for four normal subjects was 28.3 mm Hg to 29.0 mm Hg. Adair's stepwise oxygenation scheme has been used to analyze the curves with the result that a1 = 0.1514 X 10(-1) (+/- 10%) mm-1; a2 = 0.9723 X 10(-3) (+/- 8%) mm-2; a3 = 0.1703 X 10(-3) (+/- 50%) mm-3; a4 = 0.1671 X 10(-5) (+/- 2%) mm-4 for the best of four data sets. Because these constants are very sensitive to changes in the shape of the oxygenation curve, this analysis is much more useful than p50 measurements in the investigation of the various allosteric effectors of the function of hemoglobin within the red cell.


Enzymatic properties of beta-D-galactoside alpha2 leads to 6 sialytransferase from bovine colostrum.

The substrate specificity and kinetic properties of a pure sialyltransferase from bovine colostrum have been examined. The transferase appears to incorporate sialic acid into the sequence, NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAc, which is commonly found in glycoproteins. It has a strict substrate specificity for CMP-NeuAc and forms only the alpha2 leads to 6 sialyl linkage with beta-D-galactosides. N-Acetyllactosamine (Galbeta1 leads to 4GlcNAc) and asialo-glycoproteins containing the N-acetyllactosaminyl linkage at the nonreducing ends of the oligosaccharides prosthetic groups are the best acceptor substrates. Isomers of N-acetyllactosamine with beta1 leads to 3 or beta1 leads to 6 glycosidic linkages are less than 1% as effective as acceptor substates as the beta1 leads to 4-linked isomer. Lactose (Galbeta1 leads to 4Glc) is also a poor acceptor, indicating the importance of the 2-acetamido group in the N-acetylglucosaminyl residues. The unnatural substrate beta-methyl-L-arabinopyrano-side, a five-carbon analog of beta-methyl-D-galactoside which contains no 6-hydroxyl, also acts as a poor acceptor of the transferase and the sialylated product has been partially characterized. Kinetic properties of the enzyme in the presence and absence of inhibitors suggest that the transferase has an equilibrium random order mechanism.


Purification of a sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose.

CDP-hexanolamine agarose was used as an affinity adsorbent to purify a CMP-N-acetylneuraminate: beta-D-galactosyl-glycoprotein N-acetylneuraminyltransferase (EC 2.4.99.1) from bovine colostrum. Upon binding of the enzyme to the adsorbent, elution is achieved either nonspecifically, with 0.5 to 1.0 M sodium chloride, or specifically, with CDP. A highly purified sialyltransferase is obtained with a specific activity  440,000 times that of whole colostrum. Fractionation of the purified enzyme by gel filtration gives two species with different molecular weights but equal specific activities toward asialo-alpha1-acid glycoprotein (26.0 to 28.0 micronmol/min/mg of enzyme). The molecular weights of these two forms are about 56,000 and 43,000 as judged by sodium doedcyl sulfate-gel electrophoresis, sedimentation equilibrium, and gel filtration. The catalytic properties of both forms have been examined (Paulson, J. C., Rearick, J. I., and Hill, R. L. (1977) J. Biol. Chem. 252, 2363-2371). It is concluded that the lower molecular weight form may be a partially degraded species of the enzyme of higher molecular weight.


Preparation and properties of immobilized rubredoxin.

Rubredoxin, one of the three protein components of the epoxidation/hydroxylation system of Pseudomonas oleovorans was immobilized by attachment to CNBr-activated agarose (Sepharose 4B). Since this represents the first reported example of the preparation of a water-insoluble derivative of an enzyme of this type, the electron transfer and physical properties of the conjugate were examined in order to allow comparison with those of the soluble enzyme. Immobilized rubredoxin exhibits all of the major spectral properties of the soluble enzyme above 300 nm, but some distortion in the 280 nm abosrbance band was observed. The immobilized enzyme accepts electrons from dithionite or form NADPH in the presence of spinach ferredoxin-NADP reductase, and upon reduction the visible absorbance is bleached. Immobilized rubredoxin mediates the reduction of cytochrome c in the presence of NADPH and spinach reductase, although it is less efficient in this role than soluble rubredoxin. The oxidation-reduction potential of immobilized rubredoxin was determined and found to be similar to that of the soluble enzyme. In the presence of 2.5 m guanidine HCL, the immobilized enzyme is considerably more stable than soluble rubredoxin toward denaturation. After anaerobic reduction, iron was readily removed from immobilized rubredoxin by washing in 0.5 m Tris base, PH 9.5 containing 0.07 M mercaptoethanol, and the resulting immobilized apoenzyme could then be reconstituted to give back a conjugate with the original iron content, as judged from its absorbance at 497 NM. Reptition of the entire reduction-dissociation-reconstitution cycle gave the same results as were obtained after the initial reconstitution.


Action of intrinsic sialidase of rat brain synaptic membranes on membrane sialolipid and sialoprotein components in situ.

The sialo compounds in the synaptosomal membranes of young rat brain were specifically labeled in vivo by the intracranial injection of radioactive N-acetylmannosamine. More than 95% of the incorporated label was found in glycosidically bound sialic acid. Specific activities of sialic acid in the synaptic membrane gangliosides G71 (monosialo), GD1a (disialo), and GT1 (trisialo) were similar; labeling in GD1b (disialo) was consistently somewhat higher. The highest specific activity of rat brain sialidase was evenly distributed between "small myelin fragment" and synaptosomal membrane fractions, and ouabain-sensitive (Na+, K+)-ATPase also was concentrated in the latter fraction. The greatest amount of bound sialic acid was found in these subcellular fractions having the highest sialidase activity. A microsomal fraction was discovered to contain a small amount of bound sialic acid with a very high degree of radioactive labeling, but no sialidase. Release of sialic acid from the relatively intact membrane preparations by intrinsic membrane-bound sialidase occurred in two recognizable stages. There was a rapid initial release, complete within 30 min, of approximately equal amounts of lipid- and protein-bound sialic acid, corresponding to roughly half of the enzymatically releasable protein-boudn, and somewhat less than one-third of the lipid-bound, sialic acid. The remainder of the membrane sialidase-susceptible sialic acid was released in a second, slower stage. The intrinsic sialidase released 16 +/- 1% of the total sialoprotein and 31 +/- 1% of the total sialolipid sialic acid. Approximately the same amount of sialic acid is releasable from membrane sialolipid by the action of exogenous Vibrio sialidase; almost twice as much is releasable from sialoglycoprotein by this enzyme as compared with the intrinsic membrane sialidase. Each of the various membrane gangliosides appeared to be equally available to the action of the membrane sialidase. The results of this study indicate that both glycolipid- and glycoprotein-bound sialic acid in the synaptic membrane are releasable in situ by the action of the intrinsic synaptic membrane sialidase, and they suggest that this enzyme may act to modulate the physical properties of the membrane. In addition to influencing the rate of hydrolysis of endogenous membrane sialo compounds by intrinsic sialidase, pH had an effect on availability of protein-bound sialic acid. At acid pH, lipid- and protein-bound sialic acid were similarly available, but near neutral pH, gangliosides appeared to be attacked preferentially.