Influence of sodium intake on vascular and adrenal angiotensin II receptors. The restriction of sodium intake reduces the sensitivity of vascular smooth muscle to angiotensin II, but enhances its influence on the adrenal glomerulosa cell. A competitive antagonist to angiotensin II, the Sar1, Ala8 derivative (P113), which shows affinity for the same receptor as angiotensin II, was used to examine the role of receptor affinity in the effect of sodium intake on these two systems in vitro. A threshold P113 concentration reduced the response to angiotensin of aortas derived from rabbits on a high, but not on a low, salt intake. Similarly, a threshold P113 concentration inhibited aldosterone response to angiotensin in glomerulosa cells obtained from sodium-loaded, but not sodium-restricted, rats. Large doses of P113 reduced the response to angiotensin in both preparations on either diet. The competitive antagonist presumably shares affinity for the same receptor on the basis of structural similarities; thus, the results suggest that sodium restriction blunts the vasoactive effects of angiotensin by reducing the affinity of its receptor for angiotensin. In contrast, the enhanced adrenal response to angiotensin with sodium restriction cannot be explained by an altered receptor-agonist interaction, but must reside in a change in an intracellular biochemical process, sufficient to overcome the influence of reduced receptor affinity.


Reproductive hormonal function in the genetically obese (ob/ob) mouse. Reproductive function is impaired in the genetically obese (C57 B1/6J) ob/ob mouse. Serum LH, FSH, and testosterone concentrations were assessed in male ob/ob and lean littermates from 39 to 78 days of age. The lean animals demonstrated a three-fold rise in serum LH between 39 and 45 days of age that preceded a steep increase in serum testosterone which peaked at age 70 days. The obese animals did not demonstrate this LH rise; serum testosterone levels were low and had a blunted increase with age that paralleled that of normal animals. Serum FSH was lower than normal at all ages in the obese mice. The ventral prostrate and testes were small in the ob/ob mice. The castration of adult animals resulted in increased serum concentrations of both LH and FSH, with higher levels attained in the lean animals. Fifty-four-day-old castrated lean and obese mice were treated with testosterone for 15 days. Measurements of serum LH and FSH after 8 and 15 days of treatment demonstrated a marked sensitivity in the ob/ob animals to feedback inhibition of gonadotropins. This finding suggested persistent immaturity of the hypothalamic-pituitary axis in obese mice. These studies indicate that the hypogonadism of the ob/ob mouse is the result of altered hypothalamic-pituitary function.


Secretion cells in the gastrointestinal tract. The morphology and distribution of secretin (S) cells were investigated in the human and the dog. S cells were well-visualized by the indired immunofluorescence antibody technique, using a highly specific rabbit anti-secretin sera. The fluorescence reaction was not blocked by an excess amount of gastrin, cholecystokinin, glucagon, vasoactive intestinal polypeptide, or motilin, whereas secretin blocked the reaction. S cells were seen in the mucosa of the antrum and duodenum in both humans and dogs, and throughout the entire length of the canine small intestine. They were not found in the mucosa of the esophagus, fundus of the stomach, or rectum. These cells were either pyramidal in shape or pearshaped and were one-third of the size of gastrin cells. The possible significance of S-cell distribution in the antrum and small intestine is discussed.


Further study of the mechanism of thyroid hormone secretion in an in vitro model system: direct evidence for fusion of lysosomes with thyroglobulin liposomes. The interaction between thyroglobulin liposomes, as a model of colloid droplets, and thyroidal lysosomes was studied in an in vitro system. On sucrose density gradient centrifugation analysis, particulate lysosomes were distributed in a high density region and were apparently clearly separated from the liposomal peak. However, when the liposomes and lysosomes were incubated together and then analyzed, the liposomes were found to migrate with the lysosomal peak. The extent of this translocation of liposomes depended on the incubation temperature: after incubations at 37 C and 0 C, 56% and 35%, respectively, of the radioactivity of liposomes was found in the lysosomal peak. This phenomenon was inhibited by 50% in the presence of 10(-3)M chlorpromazine. Matrix-labeled liposomes [125I]thyroglobulin liposomes), and membrane labeled lipolomes ([14C]-cholesterol-labeled thyroglobulin liposomes) showed similar distribution patterns in the lysosomal peak on gradient centrifugation. The liposomal thyroglobulin was hydrolyzed in a time-dependent manner in the fraction showing both radioactivity and acid phosphatase activity. These results provide direct evidence of liposome-lysosome fusion, or formation of phagolysosomes.


Oxytocin content of microdissected areas of rat hypothalamus. Oxytocin content has been measured by radioimmunoassay in microdissected hypothalamic nuclei. Equal concentrations of oxytocin were found in the supraoptic and the paraventricular nuclei, indicating that both are major sources of the hormone. The concentration of oxytocin in the median eminence was more than three times that in either the supraoptic or the paraventricular nuclei, and significant amounts of oxytocin were also found in the arcuate nucleus and in tow anterior hypothalamic nuclei.


Postnatal development of mouse plasma and brain corticosterone levels: new findings contingent upon the use of a competitive protein-binding assay. A competitive protein-binding (CPB) assay, suitable for measuring corticosterone levels in 20 mul of mouse plasma or 100 mg of brain, is described. The postnatal development of adrenocortical function was determined in C57BL/10 and DBA/1 mice by CPB assay of basal and stressinduced levels of plasma corticosterone and resting levels of brain corticosterone. Marked increases in both basal and stressed levels of plasma corticosterone were found beginning at day 12 after birth: mean basal levels rose from about 1 mug/u99 ml on day 12 to peak values of about 10-15 mug/100 ml on days 18-20, and then declined by day 30 to the 13-day level of 2.6 mug/100 ml. This pattern differs significantly from results obtained with standard fluorometric assays for corticosterone; it was determined that a major part of this discrepancy is due to the lack of specificity of the fluorometric assay. The developmental change in brain corticosterone was similar to the pattern found in plasma. Only the stress-induced levels of plasma corticosterone showed significant genetic variation, and this did not appear until about one week after the end of the relative stress-nonresponsive period. These findings should be useful in evaluating hypotheses concerning the developmental regulation of adrenocortical function and the action of glucocorticoids in regulating the biochemical differentiation of other tissues.


A model system for studying inhibin.

A model system for studying inhibin. We report here a sensitive model for studying inhibin-like activity in crude ovine testicular extracts (CoTE). The administration of CoTE in 25-100 mg amounts to 35-day-old immature male rats, orchidectomized just prior to use, resulted in the prevention of a rise in plasma FSH levels, seen 10 h post-treatment. In a second model, CoTE was injected at 1000 h of day 35 to a group of rats that was castrated 12-24 h prior to injection, and the animals were sacrificed 6 h later; plasma FSH levels were found to be significantly suppressed. CoTE, administered subcutaneously, both in multiple doses and as a single injection, was found to be equally effective. A single injection suppressed FSH levels within 3-6 h while LH levels were unaffected. The suppressive effect was dose-dependent, reaching a maximum value at doses of 100 mg CoTE and above. FSH levels could not be suppressed below the tonic level, either by the administration of a single large dose of CoTE, or by repetitive injections of maximal doses of CoTE at 3 h intervals. The suppression in FSH levels caused by a single injection of CoTE waned with time and totally disappeared by 36h. CoTE was prepared by the heat treatment (55 C for 30 min) of an aqueous extract of ovine testis, followed by centrifugation and ether extraction of the supernatant. The aqueous layer was then dialyzed and lyophilized. The lyophilized material was found to be free of testosterone, as measured by a specific radioimmunoassay, indicating that the active factor is a heat-stable, lyophilizable, nondialyzable material, free of contamination by testosterone and probably by other steroids.


Isolation of renin granules from rat kidney cortex and evidence for an inactive form of renin (prorenin) in granules and plasma. Renin was demonstrated in particles having a sedimentation velocity similar to that of mitochondria during differential centrifugation separated renin granules from the bulk of mitochondria and lyosomes, as well as from microsomes and cytoplasm. The density of renin granules was 1.202, which differed from the mean equilibrium densities of mitochondria (1.175) and lysosomes (1.170 and 1.230) in the heavy granule fraction. In studies involving gel filtration and polyacrylamide gel electrophoreis, renin granules appeared to contain an inactive form of renin that could be activated by acid treatment, had a higher apparent molecular weight than renin, and may be a more basic molecule. Inactive renin was also studied in plasma by electrophoresis and may originate from renin granules after exocytosis by the juxtaglomerular cells. Inactive renin may be a biosynthetic precursor (prorenin) and may be activated within the cell by a specific protease consequent upon the fusion of renin granules with lysosomes, thus providing a mechanism for the rapid regulation of renin activity prior to secretion.


Hepatic glucocorticoid binders in mature and senescent C57BL/6J male mice. Because of prominent age-related changes in the responses to stress and in steroid metabolism and excretion, the effect of age on fractionated liver glucocorticoid-binding proteins was studied in C57BL/6J male mice. When cytosol, pre-labeled with [3H]corticosterone, was chromatographed on Sephadex G-100, 4 peaks were obtained. Peak 1 (excluded), peak 2 (corresponding to plasma transcortin), and peak 3 corresponding to glucocorticoid receptors isolated from nuclei) showed no significant age differences. This finding is consistent with reports that glucocorticoid-mediated induction of hepatic tyrosine aminotransferase is not altered by aging in rodents. However, there was a striking age-related decrease (80%) in peak 4 (apparent MW 29,000). Competition studies imply that peak 4 binds aldosterone, testosterone, progesterone, and corticosterone (delta4-3-keto steroids), but not estradiol or dt a plasma component. Although the function of peak 4 is not identified, the pattern of highest competition with delta4-3-keto steroids suggests that it is a steroid ring "A" reductase.


A sensitive radioenzymatic assay for dopamine, norepinephrine, and epinephrine in plasma and tissue. A double-isotope, radioenzymatic assay for measuring dopamine, norepinephrine, and epinephrine in one sample is described. The assay procedure includes incubation, solvent extraction, and thin-layer chromatography. Dopamine, norepinephrine, and epinephrine were incubated with catechol-O-methyl transferase (COMT) and [3H]S-acenosyl methionine ([3H]SAM) and were converted to the O-methylated tritiated derivatives: [3H]methoxytyramine, [3H]normetanephrine, and [3H]metanephrine, respectively. After several extraction steps the O-methylated products were purified by means of two-dimensional, thin-layer chromatography using silica gel. The thin-layer chromatographic system resulted complete separation of the three O-methylated compounds with an overlap of only 1-2%. The assay was linear from 0 to 5 ng for each catecholamine and had a sensitivity of 10-30 pg. The addition of large amounts of plasma reduced the activity of COMT, but increasing the magnesium concentration in the incubation mixture and the addition of EGTA to plasma samples improved the recoveries. Each sample was corrected for losses incurred during extraction and chromatography by using [14C]methoxytyramine, [14C]normetanephrine, and [14Ci1metanephrine that were added at the end of incubation. Several catechol compounds known to be O-methylated by COMT were examined for crossreactivity. Of the substances tested, only dihydroxyphenylalanine (DOPA) exhibited cross-reactivity. However, the apparent 30% cross-reactivity of DOPA with dopamine was due to the presence of decarboxylase activity in the COMT preparation. As little as 50 mul of trunk plasma from decapitated rats was sufficient for the determination of the three catecholamines.


Medroxyprogesterone acetate: a steroid with potent progestational activity but low receptor affinity in the guinea pig uterus. The guinea pig progestin receptor appears to be unique among mammalian receptors studied to date in that it displays a low binding affinity for some biologically potent 17 alpha-substituted progestinss. [3H]Medroxyprogesterone acetate (MPA) was synthesized and used to investigate this dichotomy between binding affinity and biological activity. The comparison of cytoplasmic and nuclear receptor binding characteristics suggested that progesterone and MPA were bound to the same receptor but with different affinities. Following an intravenous injection of 3H-steroids, the plasma level of progesterone was lower than that of MPA at all time points. Correspondingly, for up to 6 hours following steroid administration, progesterone levels were lower in uterine cytoplasm and higher in nuclei than those of MPA. However, by 24 hours, MPA nuclear levels were higher than those of progesterone, in accordance with plasma levels. We conclude that the potent biological activity of MPA relative to progesterone is due in part to its slower rate of metabolism and longer nuclear retention.


Effect of sexual maturation and androgens on prostaglandin levels in tissues of the male reproductive system in mice. Prostaglandins E and F were measured in the testis, epididymis, vas deferens, and seminal vesicles of CD-1 mice from 2 to 8 weeks of age. The concentration of PGF was higher than that of PGE in all organs studied, except for the vas deferens. The concentration of prostaglandins (PGs) was age-dependent, showing a progressive decline from immaturity to adulthood. However, in the testis, there was an apparent transient increase in the concentration of PGs in the seminal vesicle changed very little between the ages of 5 and 8 weeks. The vas deferens had a significantly higher PG concentration than any of the organs studied, and a unique pattern of changes in the levels of PGE and PGF with age. In the vas deferens of two- and three-week-old mice, the concentration of PGF was higher than the concentration of PGE, but after 4 weeks of age PGE became somewhat more abundant than PGF. Treatment of immature mice with testosterone propionate (TP) produced significant changes in PG concentrations, resulting in PG levels resembling those of adult animals. The treatment also changed the ratio of PGE to PGF in the vas deferens (from 1:2 to 1:1). Hereditary dwarf mice had higher levels of PGs in the tissues of the male reproductive system than did their normal littermates. The treatment of dwarf mice with TP generally reduced the concentration of PGs in their reproductive system and resulted in a PG pattern more characteristic of normal adult males of the same strain. The data demonstrate pronounced changes in PG levels in the tissues of the male reporductive system of mice during sexual maturation. From the present study and from previous findings, it can be concluded that these changes can be accounted for by an increase in testicular testosterone secretion.


Content of LH, FSH, and growth hormone in the pituitaries of pregnant and anestrous sheep. The total content of LH, FSH, and growth hormone was measured in pituitaries from anestrous ewes and from ewes at known stages of pregnancy. LH content was lower in the pregnant ewes than in the anestrous group. Compared with the anestrous group, a significant drop in mean LH content was seen by days 40-50 of pregnancy (894 vs 350 mug, P less than 0.001), and it reached its lowest value by 120-135 days of pregnancy (43 mug, P less than 0.001). In sheep 148 days of pregnant, mean LH content was higher than in the latter group (155 mug). The correlation coefficient between the stage of gestation and the LH content was 0.63 (P less than 0.01). There was no significant change in mean FSH content in ewes up to 135 days pregnant (range 1196-1550 mug) compared with anestrous ewes (1023 mug). In the group 148 days pregnant (531 mug), a significant decrease was seen compared with ewes at other stages of pregnancy (P less than 0.05). Stage of gestation and FSH content were poorly correlated (r = 0.07). No significant differences in growth hormone content were seen in any of the groups. The results suggest that the progressive reduction in LH response to synthetic GnRH which has been reported for pregnant sheep may be due to selective inhibition of LH biosynthesis.


A fast in vitro effect of glucocorticoids on hepatic lipolysis. A fast and direct effect of dexamethasone on liver metabolism is reported. In the perfused rat liver, addition of dexamethasone directly to the perfusate is followed by an increase in the level of free fatty acids (FFA) and glycerol. These effects of dexamethasone are evident within 30 minutes of hormone administration. The response to dexamethasone in livers from normal and adrenalectomized rats is similar. While dexamethasone and glucagon both had a lipolytic effect, these effects are not additive.


Effects of microinjection of various prostaglandins into the 3rd ventricle, median eminence and pituitary on plasma LH in rats. The effects of microinjection of PG'S (PGE1, E2, F2a) into the 3rd ventricle, median eminence (ME) and anterior pituitary on plasma LH in rats were investigated. Blood samples were obtained by jugular puncture before, and 10 and 45 min after the injection of PGS (50 or 100 mug), plasma LH was measured by radioimmunoassay. In the 3rd ventricle microinjection, PGE2 prodiced a significant rise in plasma LH. PGE1 and F2a did not significantly after plasma LH levels. In the median eminence, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. In the pituitary, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. These observations indicate that PGs act directly on the hypothalamic-pituitary axis, and that particular PG may be involved in the release of particular horomones from the hypothalamus and pituitary.


Origin of prolactin cells in the pituitary autografts in the renal capsule of young male and female rats. Origin of the prolactin cells in the pituitary grafts autotransplanted into the renal capsule was electron microscopically investigated in young male and female rats. Prolactin cells may mainly originate from the completely degranulated acidophils after grafting in both sexes. The comprehensive degranulation of acidophils may be essential condition for the development of prolactin cells. Formation of initial prolactin granules is related to the Golgi apparatus in 3 and 6 days in the males. Even prolonged transplantation for the duration of 10 and 20 days failed to develop the typical prolactin cells storing large polymorphic granules in the grafts in the males. In the females, however, the population of prolactin cells became higher (20-30%) and their granulation was more rapidly advanced than in the male. Nine and 16 days after grafting quite numerous typical prolactin cells, characterized by the pronounced vesiculation of ER stored large polymorphic granules in the females. Progressive vesiculation of ER in degranulated acidophils may be the second necessary condition for establishing the prolactin cells. Thus, the entire course of cell conversion of acidophils into prolactin cells was observed in this study. All the graft cells including prolactin cells finally turned into the cells losing the respective individuality (10 days in the males; 32 days in the females). This fine structural similarity was striking in the males. We could not, however, deny the possibility of origination of prolactin cells from the follicular cells or their adjoining undifferentiated cells.


Sterilization of female rats by neonatal placement of estradiol micropellets in anterior hypothalamus. A pair of micropellets of a 1% or 10% estradiol (E2)-paraffin mixture (containing 0.2 or 2 mug E2, respectively) or paraffin alone were implanted subcutaneously or intracerebrally in 5-day-old female rats. Animals given 10% E2 pellets became sterilized regardless of loci of the pellets. Of those which had received 1% E2 pellets, only animals with micropellets in the anterior hypothalamus became sterilized. It is suggested that neuronal components which are affected irreversibly by neonatal estrogen treatment are localized in the anterior hypothalamus.


Alterations of insulin-secreting response to glucose in human infants during the early postnatal period. Oral glucose tolerance tests were performed in healthy infants, aged one to 29 days. Capillary blood samples were obtained from heel stabs for estimation of glucose and insulin immediately before and 30, 60 and 120 minutes after the administration of glucose (2.0g per kg body weight). The younger infants tended to have delayed and diminished insulin responses to a glucose load than did older infants. The ratio of the increment of insulin concentration to the increment of glucose concentration at 30 minutes following a glucose load in younger infants, aged one to 20 days, was below 0.4. The ratio in infants older than 20 days was above 0.5. From these results it is evident that  the pancreas of human infants begins to respond to the stimulation by glucose during the early postnatal period and this response becomes even obvious after 20 days of age.


In vitro biosynthesis of human chorionic follicle stimulating hormone. In order to explore the possibility that human chorionic FSH (hCFSH) may be synthesized in vitro by the placenta and secreted into the culture media, chorionic tissue of the first trimester was cultivated in the radioactive medium prepared byadding 3H-proline and/or 14C-glutamic acid. Purification of biosynthesized hCFSH from the media was carried out by a combination of Sephadex G-100 gel filtration, DEAE-cellulose chromatography and polyacrylamide disc-gel electrophoresis...


Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin. Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.


Differential effects of glucagon on induction of rat liver glucose-6-phosphate dehydrogenase by liver injury and dietary change. Glucagon effectively prevented the increase in glucose-6-phosphate dehodrogenase activity of rat liver following the administration of a glucose-casein mixture without altering the amount of the diet consumed. However, the increase of the enzyme level in carbon tetrachloride-injured rat liver was virtually insensitive to glucagon. The results obtained gave further evidence for the difference between these two induction mechanisms of glucose-6-phosphate dehydrogenase.


Serum enolase and pyruvate kinase activities in the diagnosis of myocardial infarction. Sequential determinations of serum enolase and pyruvate kinase (PK) activities were carried out in addition to routine enzyme assays on 191 patients admitted to a coronary care unit. The upper reference values established in 61 healthy laboratory staff members were 21 U/1 for enolase and 76 U/1 for PK. Of 99 patients with a confirmed diagnosis of myocardial infarction (MI), 97 manifested raised serum enolase during the period of observation; 85 of these patients had raised serum PK. The incidence of raised values for serum PK among patients considered not to have sustained infarction was similar to that shown by the routine enzymes, but serum enolase activity was much more frequently raised in these patients. The time-course of elevation of serum enolase and also that of PK are discussed in relation to the changes in other serum enzymes routinely measured for MI.


Red cell metabolism in high and low glutathione goats. The following biochemical features were measured in the red blood cells of high and low glutathione goats: reduced glutathione (GSH) stability; GSH regeneration; glucose consumption; lactate production; levels of adenosine triphosphate and 2,3-diphosphoglycerate, and the activities of nine different enzymes of Embden-Meyerhof and pentose phosphate pathways of glucose metabolism. Apart from significant differences in the GSH stability the results of all the tests were found to be similar in the two groups.


Effect of ethanol and zinc on ALA-dehydratase activity in red blood cells. Subcutaneous injection of zinc as sulphate to rats activates sigma-aminolaevulinic acid dehydratase (ALA-D) in red blood cells within few days of injection. This activation counteracts the inhibitory effect of orally administered ethanol.


Phenacetin and the liver. The influence of phenacetin in acute and chronic doses on membrane-bound mitochondrial enzymes in the rat. Following the administration of phenacetin in single and in multiple high doses, enzymes bound to the inner mitochondrial membrane of the liver were determined. Acute doses of phenacetin (75% of oral LD50) failed to produce any effect. The chronic administration of phenacetin provoked a small but statistically significant decrease in the TD-trnashydrogenase activity. This observation indicates that liver damage may occur in patients with phenacetin abuse.


The structure of kinetoplast DNA. 1. The mini-circles of Crithidia lucilae are heterogeneous in base sequence. We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia  luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.


Translation of the mRNA for rabbit uteroglobin in cell-free systems. Evidence for a precursor protein. Uteroglobin, an hormonally induced protein composed of two similar subunits, represents around 50% of the proteins synthesized and secreted into the uterine lumen of rabbits treated sequentially with estradiol and progesterone. The endometrium of these animals was used as a source for the isolation of the mRNA for uteroglobin. Poly(A)-rich RNA, extracted from purified polysomes with phenol chloroform and isolated on oligo(dT)-cellulose columns, contains one fourth of the total protein coding activity of the endometrium. Between 20--25% of the polypeptides synthesized by this RNA in cell-free systems derived from Krebs II ascites cells or wheat germs react with a monospecific antiserum prepared in guinea pigs against uteroglobin. The material bound to the antibody was identified as a precursor of uteroglobin according to the following criteria. 1. The product synthesized in vitro can be displaced from the complex with the specific immunoglobulin by purified uteroglobin. 2. Analysis of the immunoprecipitate on polyacrylamide gels containing urea and dodecylsulfate demonstrate the existence of a single labelled polypeptide with an apparent molecular weight larger than the uteroglobin subunits. 3. The tryptic digest of this polypeptide, labelled in vitro with [3H]lysine, shares seven peptides with mature uteroglobin labelled with the same amino acid in perfused uteri, and exhibits and additional peptide not present in uteroglobin. 4. Injection of the same mRNA preparation into Xenopus oocytes results in the production of uteroglobin. The endometrium of intact animals treated with estradiol alone also contains the same mRNA bound to polysomes but in a smaller proportion, indicating that the progesterone-induced synthesis of uteroglobin is accompanied by an accumulation of the specific mRNA in the polysomes.


The structure of kinetoplast DNA. 2. Characterization of a novel component of high complexity present in the kinetoplast DNA network of Crithidia luciliae. 1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.


The incorporation of newly synthesized RNA into nuclear ribonucleoprotein particles after oestrogen administration to immature rats. Ribonucleoprotein particles, known as informofers or heterogeneous nuclear ribonucleoprotein particles (hnRNA - protein), have been extracted from rat uterine nuclei and found to have properties similar to those characterized from other tissues. Incorporation of RNA precursors into the RNA of these particles is stimulated up to 8-fold by oestrogen administration to rats. When uteri are dissected from oestrogen-treated rats and incubated in vitro with radioactive RNA precursor, only the RNA in the ribonucleoprotein particles is synthesized at a rate faster than can be accounted for by increase in the uptake of precursor. This contrasts with previous studies where both hormone treatment an incorporation of radioactive precursor were performed in vivo and the synthesis of all RNA species was stimulated [Knowler, J.T. and Smellie, R.M.S. (1971) Biochem. J. 125, 605--614; Knowler, J.T. and Smellie, R.M.S. (1973) Biochem. J. 131, 689--697].


Simultaneous translation of structural and nonstructural proteins from Semliki-forest-virus RNA in two eukaryotic systems in vitro. The Semliki Forest virus genome, 42-S RNA, and the virus-specific intracellular 26-S RNA were translated in two cell-free protein-synthesising systems, the wheat germ extract, and a partially purified system from mammalian tissues. The 26-S RNA directed the synthesis of structual proteins only, as revealed by tryptic peptide mapping. About 75--80% of the radioactivity in the products comigrated with capsid and about 4--8% with envelope protein peptides. All the capsid peptides and the full-sized capsid protein were found in the products in vitro, no complete envelope protein was formed and fewer than half of the envelope peptides were detected. This result is consistent with reports that there is only one initiation site for the translation of virus structural proteins, and that the capsid protein is N-terminal in the polyprotein followed by envelope proteins. The systems programmed with 42-S RNA yielded virtually the same structural peptides. However, the bulk of the radioactivity was in peptides which did not comigrate with the structural ones. These peptides were mostly associated with relatively small-sized products. This shows that Semliki Forest virus 42-S RNA has at least two initiation sites, one for the structural proteins and the other(s) for the nonstructural proteins.


The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole. The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.


Snake venom toxins. The amino-acid sequences of three toxins (CM-8, CM-11 and CM-13a) from Naja haje annulifera (Egyptian cobra) venom. Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.


The enzyme complex citramalate lyase from Clostridium tetanomorphum. 1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.


Acetic anhydride: an intermediate analogue in the acyl-exchange reaction of citramalate lyase. 1. Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzyme-anhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride. 2. A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S-citramalate appears to be non-competitive with iodoacetate. 3. The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lyase. 4. The formation of 6-citryl thiolester from the 1-thiolester via the cyclic citric anhydride provides a chemicla model for enzymic acyl exchange. 5. The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters.


Studies on the role of uncharged tRNA in pleiotypic response of animal cells. Experiments were carried out to assess the physiological significance of the charging level of tRNA. Histidinol, a competitve inhibitor of charging of tRNAHis, was used to induce uncharged tRNA in mammalian cells. It is demonstrated that both in the presence of histidinol and under histidine depletion about 40% of the tRNAHis is uncharged. Concomitant with this appearance of uncharged tRNA(a) the pools of GTP and ATP are decreased rapidly by 25--30%; (b) the synthesis of both protein and ribosomal RNA is inhibited, whereas that of nucleoplasmic RNA is not affected; (c) the uptake of 2-deoxyglucose, phosphate, Ca2+; uridine and adenosine is inhibited; and (d) the growth of 3T6 fibroblasts is arrested. It is suggested that the appearence of uncharged tRNA is one of the earliest events occurring under conditions of amino acid starvation, which in turn causes the various metabolic changes observed.


On the mechanism of malonyl-CoA-independent fatty-acid synthesis. Different properties of the mitochondrial chain elongation and enoylCoA reductase in various tissues. 1. NADPH-specific mitochondrial enoyl-CoA reductase can be assayed by a sensitive radioactive test, employing tritium-labelled NADPH, synthesized in a prefixed reaction from D-[1-3H]-glucose via the hexokinase and glucose-6-phosphate dehydrogenase reactions. 2. Liver, kidney cortex, heart muscle, skeletal muscle, brown adipose tissue, brain cortex, and aortic intimal tissue are investigated concerning chain lengths specificity of the chain elongation and the enoyl-CoA reductase. Medium-chain acyl-CoA compounds prove to be the best primers for the chain elongation. Enoyl-CoA reductases still show large incorporation rates with hexadecenoyl-CoA. 3. The differences in the chain lengths specificity of the chain elongation and enoyl-CoA reductase can be explained by the inhibitory effect of long-chain acyl-CoA derivatives on the 3-hydroxyacyl-CoA dehydrogenase. 4. The nucleotide specificity in the different tissues reveals two types of chain elongation: In addition to liver and kidney cortex, mitochondria of brown adipose tissue need NADH + NADPH for optimal chain elongation, whereas heart muscle, skeletal muscle and aortic intimal mitochondria only need NADH. 5. Different physiological roles are proposed for the two types. The "heart type" may be of importance in the conservation of reducing equivalents or acetate units in the anaerobic state, the "liver type" may play a role in the transfer of hydrogen from NADPH to the respiratory chain. In addition, the mitochondrial chain elongation may serve as bypass of the first part of the respiratory chain.


The binding of tritiated elongation-factors 1 and 2 to ribosomes from Krebs II mouse ascites-tumore cells. The influence of various antibiotics and toxins. The effect of a number of different antibiotics and toxins on the capacity of Krebs II mouse ascites ribosomes to bind 3H-labelled elongation factors (EF-1 and EF-2) has been examined. It was found that abrin and ricin inhibit the binding of EF-2, while diphtheria toxin, sparsomycin, streptovitacin A, and cycloheximide had essentially no effect on its binding. Of the other compounds examined, sparsomycin was unique in its capacity, under some circumstances, to significantly affect the binding of aminoacyl-tRNA and EF-1 to ribosomes. Fusidic acid appears to nonspecifically enhance the binding of both EF-1 and EF-2.


Kinetic isotope effects on the catalytic activity of pig-plasma benzylamine oxidase. 1. Isotope effects on the catalytic activity of benzylamine oxidase at pH 7 and 9 have been studied by steady-state and transient-state kinetics methods, using [alpha,alpha-2H]benzylamine as the substrate. 2. Replacement of the alpha-hydrogen atoms in benzylamine by deuterium has no significant effect on substrate-binding to benzylamine oxidase, neither does it affect the rate of reoxidation of the reduced form of the enzyme. Conversion of the primarily formed enzyme-substrate complex into the reduced enzyme species, however, exhibits an isotope effect of about 3. 3. The data obtained are consistent with a mechanism in which reduction of benzylamine oxidase takes place by a rapid pre-equilibration between enzyme and substrate to form an amine-pyridoxal Schiff-base, which is then tautomerized by a comparatively slow prototropic shift to an amino aldehyde-pyridoxamine Schiff-base from which there is a rapid hydrolytic release of the aldehyde product corresponding to the amine substrate. Proton abstraction from the alpha-carbon of the amine moiety in the primary Schiff-base appears to be at least partially rate-limiting for the tautomerization step, and hence for the entire process of enzyme reduction.


Action of thrombin on ovine, bovine and human pituitary growth hormones. 1. This communication reports the action of bovine thrombin on ovine, bovine and human growth hormones. Thrombin cleavage was shown to be restricted to a single homologous peptide bond in all three growth hormones (at sequence positions 133--134 of the ovine and bovine hormones). 2. Ovine growth hormone was the most sensitive to the action of thrombin, bovine growth hormone was attacked to a relatively less extent, and human growth hormone was the most resistant to the enzyme. 3. After reduction and carbamidomethylation of the disulfide bonds in thrombin modified ovine growth hormone, the two fragments (residues 1--133 and 134--191) were isolated. The large NH2-terminal thrombin fragment of the hormone (residues 1--133) was found to be inactive in the rat tibia test, whereas a tryptic fragment (residues 96--133) isolated in an independent way gave measurable responses.


The use of metrizamide as a density-gradient medium in studies of rat-liver polysomes. The behaviour of rat liver cytoplasmic ribonucleoproteins in metrizamide has been studied to determine whether the iodinated compound would offer any advantage over other centrifugation media for studies of polysome structure and function. 1. Polysomes had a density of 1.295--1.300 g/cm3 in metrizamide gradients which was also the density of glycogen. However, the polysaccharide reached its equilibrium more rapidly than the polysomes. Thus a short centrifugation of a polysome suspension from non-starved rats over a 40% metrizamide cushion was sufficient to eliminate more than 85% of the glycogen with a polysome yield of about 75%. 2. Ribosomal subunits had neighbouring densities (1.23 and 1.20 g/cm3 for large and small EDTA-derived subunits; 1.23 and 1.21 g/cm3 for large and small KCl/puromycin-derived subunits, respectively). Polysomal messenger ribonucleoproteins were heterogeneously distributed (phi = 1.12 to 1.23 g/cm3) and overlapped with subunits in a similar manner as in sucrose gradients. 3. Analysis of a post-mitochondrial supernatant in metrizamide showed a clear separation of free polysomes, rough membranes and the soluble phase.


Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix. The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.


Immunochemical detection of the Thomsen-Friedenreich antigen (T-antigen) on the pig lymphocyte plasma membrane. A method for the isolation of lymphocytes from pig peripheral blood and preparation of plasma membranes was developed. The method resulted in a ten-fold increase in 5'-nucleotidase activity and neuraminic acid, relative to protein. A cholesterol/phospholipid ratio of 0.93 was obtained. Alkaline borohydride treatment of isolated plasma membrane, after desialylation, released the disaccharide beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol (the immunodominant group of the T-antigen), which was identified by gas chromatography using a crystalline standard. The disaccharide was not found without prior desialylation, indicating that all the disaccharide units were substituted by neuraminic acid. Serological evidence using the agglutinins from Arachis hypogoea and Helix pomatia confirmed the presence of this disaccharide (T-antigen) in the lymphocyte membrane and indicated that it was the major alkali-labile oligosaccharide of the pig lymphocyte membrane.


Interaction between membrane functions and protein synthesis in reticulocytes. A membrane constituent which inhibits protein synthesis in cell-free system. The reticulocyte cell membrane was investigated for the role that it plays in cellular protein synthesis. It was found that a strong inhibition in protein synthesis occurred when a membranal extract was added to a cell-free protein-synthesizing system. The membranal extract was obtained by disrupting and then sulubilizing cell membranes with the non-ionic detergent Triton X-100. The inhibition in protein synthesis could neither be attributed to an RNAse-type action nor to a reduction in the energy level of the system. Preliminary results show that the principal site of action occurs at the elongation or/and termination stages of protein synthesis. Initial purification of the inhibitory component was achieved.


Determination of molecular weight and molecular structure of rat-liver pyruvate carboxylase. The molecular weight of pyruvate carboxylase isolated from pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and electron microscopy. The enzyme molecule appeared as a tetramer with the four subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a molecular weight Mc=0r of 280 000 and an So20,w of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4 degrees C or incubation at -- 20 degrees C at low protein concentrations. The isolated trimers and dimers had a molecular weight Mc=0r of 210 000 and 140 000, respectively, and an So20,w of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20 degrees C yielded enzymically inactive subunits (Mc=0r = 70 000; So20,w = 4.95 S). The molecular weights (for pyruvate carboxylase and its subunits), as calculated from the subunit diameter observed in the electron microscope, were consistent with the values obtained from sedimentation studies.


Absolute configuration of 3-hydroxy fatty acids present in lipopolysaccharides from various bacterial groups. The absolute configuration of 3-hydroxy fatty acids has been studied, which are present in the lipopolysaccharides of the following bacteria: Phodopseudomonas gelatinosa, Rh. viridis, Rhodospirillum tenue, Chromobacterium violaceum, Pseudomonas aeruginosa, Bordetella pertussis, Vibrio metchnikovii, Vibrio cholerae, Salmonella spp., Escherichia coli, Shigella flexneri, Proteus mirabilis, Yersinia enterocolitica and Fusobacterium nucleatum. The 3-hydroxy acids were liberated by strong alkaline hydrolysis, converted to 3-methoxy acid L-phenylethylamides and analyzed by gas-liquid chromatography. With the aid of authentic D-3-hydroxy fatty acids it was shown for all lipopolysaccharides that the 3-hydroxy acids, regardless of chain lengths, branching, 3-O-substitution or type of linkage, possess the D-configuration. 2-Hydroxydodecanoic acid, which is present in some lipopolysaccharides, was analyzed in an analogous way and shown to possess the L-configuration.


Protective effect of elongation factor 2 on the inactivation of ribosomes by the toxic lectins abrin and ricin. The inactivation of rabbit reticulocyte ribosomes by abrin and ricin A-chains was studied by incubating ribosomes with the A-chains and testing, after various periods of time, aliquots of the ribosomes for their ability to polymerize phenylalanine. The presence of elongation factor 2 (EF-2) reduced the rate of inactivation of ribosomes by the A-chains. The protective effect of EF-2 was strongly enhanced by GTP and, to a lesser extent, also by GDP or dGTP. Other nucleotides had no demonstrable effect. Much less protection was found after binding of Phe-tRNA to ribosomes in the presence of EF-1 (enzymic binding) or in the presence of high Mg2+ concentration (non-enzymic binding). The data indicate that when EF-2 binds to the ribosomes it completely or partially covers the target site for abrin and ricin A-chains. The possibility that EF-1 also binds to this site is discussed.


Variable stabilities and recoveries of rat-liver RNA polymerases A and B according to growth status of the tissue. 1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed.


Large-scale purification and some properties of the mitochondrial aspartate aminotransferase from pig heart. A method has been developed which allows isolation of 0.3--0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.


Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells. Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.


Coordinated action of pectinesterase and polygalacturonate lyase complex of Clostridium multifermentans. The polygalacturonate lyase and pectinesterase activities of Clostridium multifermentans, both produced extracellularly when the organism grows on pectin or polygalacturonate, have been suggested to be associated in a single complex. Both enzymic sites act on their respective substrates by single-chain action patterns, as shown by equivalent release of terminal tritium label and total product throughout the reaction. From these results, the Km and V of the lyase, and the amount of lyase activity present, we calculate the steady-state concentration of lyase substrate expected during action of the two sites on pectin if the sites are independent. No such steady-state concentration of lyase substrate was observed. Therefore, we conclude that the two types of active site act in a coordinated manner; the polysaccharide chain passes from the esterase site to the lyase site without intermediate dissociation and rebinding. This 'molecular disassembly line' constituted by the two sites may represent a system of general significance in synthesis and degradation of biological polymers.


Hepatic 15-hydroxylation of corticosteroids in the rat. Substrate specificity studied in the isolated perfused liver. The substrate specificity of a sex-specific hepatic 15-hydroxylase active on different C21O2 and C21O3 steroids was studied in the isolated perfused liver from female rats. Liquid-chromatographic separation methods in combination with computerized gas chromatography-mass spectrometry was employed to identify the metabolites formed. The majority (between 75-90%) of 15-hydroxylated compounds isolated were present as monosulphate conjugates while smaller amounts of disulphates were also detected. Hydroxylation was found to take place exclusively at position 15 beta. A certain number of 11-deoxy-21-hydroxy, 11-oxo-21-hydroxy and 11,21-dihydroxy steroids with a 3-keto-delta4-, 3-keto-5 alpha(5 beta)-, 3alpha, 5alpha- or 3 beta, 5 beta-structure were readily converted to 15 beta-hydroxylated metabolites. Depending on the structure of the substrate, between 20 and 87% of the total metabolites formed were 15 beta-hydroxylated. 5 alpha-Reduced steroids were better substrates for the hydroxylase than the corresponding 3-keto-delta4- or 5 beta-reduced compounds. The configuration of the hydroxylgroup at C-3 did not affect the degree of 15-hydroxylation. 11 beta-Hydroxylated steroids served as better substrates than the corresponding 11-dehydro-, 11-deoxy- or 11 alpha-hydroxy compounds. 5 alpha-Dihydrocorticosterone and 3 alpha, 5 alpha-tetrahydrocorticosterone were the best substrates for the 15 beta-hydroxylase.


The complete amino-acid sequence of rat thyrocalcitonin. The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The sequence determined was H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly-Ala-Pro-NH2. This sequence differs in only two positions from that found in the human hormone, i.e. leucine-16 in the rat vs phenylalanine-16 in the human, and serine-26 in the rat vs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.


Partial characterization of hog renin purified by affinity chromatography. A method has been set up to purify renin on a large scale by affinity chromatography using Pepstatin, a potent inhibitor of renin, as a ligand. Pepstatin was covalently coupled to Sepharose via six different spacer 'arms'. The Sepharose-hexamethylenediamino-Pepstatin appeared to be the better derivative for renin purification even at a concentration as low as 160 nmol of Pepstatin/ml of moist gel. Renin was extracted from 100 kg of hog kidneys and semi-purified by ammonium sulfate precipitations and chromatography on DEAE-cellulose. The active fraction (48.5 g of proteins) was applied on a 500-ml affinity column. Renin was eluted in the starting buffer containing 6 M urea. Renin was purified 120-fold by the affinity chromatography step with a 79% recovery. Physico-chemical characterization of highly purified renin was performed. Isoelectrofocusing on a pH gradient from 3 to 6 showed a major peak with an isoelectric point (pI) of 4.95 and a minor peak (pI = 4.70). Polyacrylamide gel electrophoresis, pH 7.8, at different gel concentrations, showed a single peak of renin activity which was found in the major protein band. Molecular size estimated on agarose-acrylamide gel filtration was 40 000. All these physical parameters were similar before and after purification.


Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay. 1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.


Glutamine transaminase from brain tissue. Further studies on kinetic properties and specificity of the enzyme. Glutamine transaminase, highly purified from rat brain, was studied. In the first series of experiments, the kinetics of the transamination reaction between 2-oxoglutaramate and phenylalanine were examined in order to determine the type of reaction mechanism. This proved to be of the ping-pont type, as can be expected for a transamination. The specificity of the enzyme for various amino acids and 2-oxo acids was then studied in detail. The most active substrates were glutamine, methionine and ethionine as amino-group donors, and phenylpyruvate, glyoxalate and 2-oxo-4-methiobutyrate as amino-group acceptors. For these and several other substrates, the kinetic constants, V and Km, were determined.


Complementation of subunits from glycogen phosphorylases of frog and rabbit skeletal muscle and rabbit liver. Activity can be induced in potentially active rabbit skeletal muscle phosphorylase monomers covalently bound to Sepharose by noncovalent interaction with soluble subunits carrying inactive pyridoxal 5'-phosphate analogs or even salicyladlehyde. These analogs are themselves incapable of reconstituting active holophorphorylase from apophosphorylase. Phosphorylases with one intrinsically inactive and one potentially active subunit have about one half of the activity of the native phosphorylase dimer. The usefulness of this technique for subunit complementation was demonstrated by forming hybrid phosphorylases with inactive Sepharose-bound rabbit skeletal muscle subunits containing pyridoxal 5'-phosphate monomethylester and soluble activatable frog muscle and rabbit liver phosphorylase monomers. The inactive Sepharose-bound subunit induced in each case activity in the soluble subunit. But whereas the inactive rabbit muscle phosphorylase subunit even transmitted its characteristic temperature dependence of the rate of the reaction to the frog muscle subunit, it could not propagate its control properties to the liver enzyme. Differences of hybrid phosphorylases are related to immunological and amino acid divergencies among the component enzymes.


Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors. The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.


Acrodermatitis enteropathica: recent findings concerning clinical features, pathogenesis, diagnosis and therapy. Acrodermatitis enteropathica (AE) was diagnosed in 2 siblings, boy and girl, at the age of 10 and 6 weeks. The family history is unremarkable except for consanguinity 5 generations previously. The clinical symptoms of the 2 patients conformed to the known features of AE, the gastrointestinal involvement loosing its significance with increasing age. In one patient in a stage of exacerbation the serum level of oleic acid (18:1) was lowered and of linoleic (18:2) acid slightly increased while that of arachidonic acid was decreased (Fig. 4). In both patients the serum zinc levels were significantly lowered. Under substitution with ZnSO4 the clinical condition improved and the serum zinc levels returned to normal. Histologically the small bowel mucosa was practically normal. Ultrastructural examination of jejunal biopsies revealed rather unspecific changes in the enterocytes in the form of numerous multivesicular bodies. The Paneth cells sometimes contained irregularly formed inhomogeneous structures within their cytoplasm. In addition the secretory granules varied in size and displayed a granular heteromorphic matrix. Frequently they were confluent and formed giant granules.


[Intestinal disaccharidase and alkaline phosphatase activities of jejunal biopsies in small bowel diseases of children (author's transl)]. Intestinal disaccharidase activities were determined in 294 jejunal biopsies obtained from 254 children with various disorders of the small bowel, and alkaline phosphatase activity was measured in 251 biopsies. In normal mucosa a broad range of enzyme activity was found corresponding with the data in the literature. A primary disaccharidase deficiency was observed in 5 children with congenital sucrase-isomaltase deficiency and in a 12-year-old Egyptian boy with acquired lactase deficiency. A secondary generalized depression of disaccharidase activity and a diminution of alkaline phosphatase activity existed chiefly in patients who had severe or moderate mucosal damage, also in active coeliac disease and during gluten loading, in protracted diarrhoea of infancy, chronic malabsorption of unknown origin and agammaglobulinemia. During remissions enzyme activities recovered together with mucosal improvement. Low levels of enzyme activities were also seen in some cases of protracted diarrhoea of infancy and chronic malabsorption of unknown origin although only mild mucosal lesions were demonstrated.


[Tryptic and chymotryptic activity in the faeces of children of different age groups (author's transl)]. Measurement of tryptic and chymotryptic activity in the faeces was not disturbed by bacterial proteolytic activity of different bacteria such as proteus, pseudomonas, coli, enterococci, bacteroides. Both activities within a group follow a logarithmic normal distribution. Lower limit of the standard deviation is 51% upper limit 129% for tryptic activity, respectively 60 and 170% for chymotryptic activity. There were no differences in chymotryptic activity between the 10 age groups comprising 157 healthy children, whereas a significant difference could be found for tryptic activity between premature and older children. Daily fluctuations of the enzyme activities are quite high in the same individual, and only reduced in "bottlefed" infants with constant nutrition. In prematures and very young infants chymotryptic activity predominates, later tryptic activity. Influence of increased and decreased bowel movements on deviation of the data was tested. There was, however, no real alteration of enzyme activity due to the bowel dysfunction beyond the standard deviation of the control groups. But passage time and nutrition have to be considered beside other factors in the wide distribution of the enzyme activities and the latter limits the value of this method.


[Leucine sensitive hypoglycemia. Follow-up studies under treatment with diazoxide (author's transl)]. Since 1969 leucine sensitive hypoglycemia has been diagnosed in 4 infants. Elevated serum insulin levels suggested hyperinsulinism; in 1 infant islet cell hyperplasia was demonstrated by morphological examination of pancreatic tissue. All cases were treated with diazoxide and have been followed under therapy for periods ranging from 4 months to 6 1/4 years. The upper limit of dosage was 15 mg/kg. The blood glucose values promptly became normal in 2 infants under this regimen. The development of these patients was normal. In the 2 other infants treatment was effective only after the additional restriction of daily protein intake to 2-2.5 g/kg. Severe cerebral damage occurred in one of these infants due to recurrent episodes of hypoglycemia, causing further development to be considerably restricted; the other one developed satisfactorily but with moderate mental retardation. Both children are suffering from cerebral convulsions. In all patients hypertrichosis was the only side effect.


[HGH secretion after oral application of L-dopa and L-carbidopa (author's transl)]. The stimulatory effect of L-Dopa and L-Carbidopa (Nacom) on HGH secretion was determined in 12 children of normal height aged from 6 to 14 years. Each child received a standard dose of 250 mg L-Dopa and 25 mg L-Carbidopa p.o. HGH concentration in the serum was determined at standard intervals. All subjects showed a sufficient increase of HGH. The mean value was 19.6 ng/ml. According to the maximum values of the HGH concentration the sample can be divided into two groups; the first group reached the highest values after 20--40 min, the second one after 60--90 min. On evaluation of the curve of the mean values it appears that 2 blood samples taken 40 and 90 min after the ingestion of L-Dopa and L-Carbidopa are sufficient in order to exclude HGH deficiency. The theoretical background for HGH secretion after stimulation by L-Dopa and L-Carbidopa is discussed.


Insulin antibodies in 104 children with diabetes mellitus. Binding capacity for porcine, bovine, and human insulin was estimated in 104 diabetic children using homologous systems of iodinated and noniodinated insulin. All patient sera bound porcine and bovine insulin whilst 11.5% did not bind human insulin. There was no clear correlation between duration of insulin treatment and binding capacity. However, some patients with only short duration of treatment had high capacity for porcine insulin. The binding capacity for human insulin was low in most patients. High insulin requirement was on average combined with high binding for bovine insulin. Cases of diabetes, which are difficult to control, treated with individual mixtures of porcine and bovine insulin, often showed high binding for porcine and also for human insuline. The value of estimations of insulin-binding capacity in diabetic children is debatable. However, the high incidence of antibodies seems to justify the use of low immunogenic (monocomponent) insulin preparations.


[On the validity of W170 as a measure of physical performance capacity in the assessment of children with heart disease (author's transl)]. W170, i.e., the work rate at a heart frequency of 170 beats per minute, and maximal oxygen uptake were determined in one performance in 20 boys and 23 girls from 6 to 16 years of age on a bicycle ergometer. The results were related to normal values according to age and height. In different age and sex groups correlation coefficients between W170 and maximal oxygen uptake amounted to values between 0.36 and 0.66. Accordingly only 13--44% of the variance of maximal oxygen uptake were covered by W170. The imperfect conformity between indirectly and directly measured cardiovascular performance capacity was due to the considerable variability of the maximal heart rate. Accordingly we found correlation coefficients from 0.28 to 0.69 for the relations between maximal heart rate on the one hand and the difference between percentage values of maximal oxygen uptake and W170 on the other hand. That means that the error of an indirect determination by means of the W170 is mostly higher, the more the maximal heart rate differs from the mean value of the group. The estimation of cardiovascular performance capacity by means of the W170 yielded higher relative values than by means of maximal oxygen uptake. The reason for this is probably that in our present investigations we used another type of ergometer than that we had used earlier for determination of normal values of W170, to which we referred in our present investigations. We suggest that the different gyrating mass of different types of ergometers in spite of identical calibration may influence the results via a different mechanical efficiency.


Increased intracranial pressure in so-called 'normal-pressure hydrocephalus'. Five patients exhibited an apparently communicating hydrocephalus -- type of disorder, with ventricular reflux on the cisternograms, lack of air-encephalographic parasagittal air-filling and clearly enlarged temporal horns. The group seemed heterogeneous, with regard to age distribution; possible etiological factors and duration of the hydrocephalic process. In all 5 patients increased intracranial/intrathecal pressure was observed: in 2 patients, only with intrathecal measurement in the lumbar area, but in the remaining 3 patients with epidural/ventricular fluid pressure monitoring. Continuously increased pressure extending over 1 or more days was observed in these patients. This pressure increase may seem different from the episodic pressure increase previously reported in normal-pressure hydrocephalus. These patients seem to be cases of so-called normal-pressure hydrocephalus according to the commonly accepted criteria. It is therefore suggested that the term normal-pressure hydrocephalus should be abandoned, and replaced by, e.g., low-pressure hydrocephalus, malresorptive or malabsorptive hydrocephalus.


Brain tumors simulating purulent meningitis. Three cases of primary brain tumors simulating purulent meningitis have been described. Two patients were infants suffering from ependymoma of the posterior fossa, while the third was a 35-year-old man with astrocytoma of the temporal lobe. All cases were characterized by acute onset with fever, signs of meningeal irritation without any other neurological signs, and marked CSF pleocytosis. The diagnosis in 2 cases was made only at necropsy, and the third case was correctly diagnosed only after a delay. The possible occurrence of brain tumor and meningitis simultaneously was considered and seemed unlikely in our cases. A possible explanation for the clinical and CSF findings was irritation of the leptomeninges by the tumor and its breakdown products.


Progressive multifocal leucoencephalopathy and Rosenthal fibres. The present paper is the study of a case of progressive multifocal leucoencephalopathy that appeared in the course of a malignant reticulosis accompanied by paraproteinosis. Due to the lesions in the brain stem and cerebellum, the case evolved clinically as a pure cerebellar form. The particularity of the morphologic aspect, not as yet described, was the abundant presence of Rosenthal fibres in the older demyelination foci. These fibres exhibited a typical structure both in the electron and optic microscope. Emphasis is laid on the connection between the formation of Rosenthal fibres and blastic cellular transformation of the glial system.


Absence of relationships between L-dopa plasma levels and therapeutic effect in Parkinson's disease treated with L-dopa. The plasma concentration of dopa was studied in 15 parkinsonian patients after a single administration of L-dopa. The study was undertaken in order to determine if correlation between plasma dopa concentration and clinical performance as a result of the tests carried out before and after a single dose of dopa, was present. Our data confirm the absolute absence of correlation between dopa levels in plasma and clinical improvement during long-term treatment. Moreover, clinical responses to the performance tests were not related to the concentration of circulating L-dopa in the plasma. Therefore dopa levels cannot be useful in clinical practice but only for the prevention of too high concentrations of plasma L-dopa which often cause a few side-effects.


Effects of long-term L-dopa therapy on carbohydrate metabolism in patients with Parkinson's disease. Carbohydrate metabolism and insulin secretion were investigated in 26 patients with Parkinson's disease before and during L-dopa treatment. Oral glucose tolerance tests were performed on 13 patients treated with L-dopa alone and on 7 patients treated with L-dopa combined with Carbidopa. Intravenous glucose tolerance tests were performed on additional 6 patients treated with L-dopa alone. Results indicate that chronic L-dopa administration does not modify glucose metabolism. It was observed only a significant decrease of insulin secretion after oral glucose in the early phase (15th day) of treatment with L-dopa alone. This temporary effect may be related to the peripheral conversion of L-dopa to dopamine since insulin secretion during combined therapy was normal. The mechanism by which L-dopa transiently inhibits insulin release is not clear. Perhaps the oral administration of L-dopa alone causes an alteration in some gastrointestinal functions which are involved in the handling of oral glucose.


Sarcoidosis with incomplete bilateral bundle branch block pattern disappearing following steroid therapy: an electrophysiological study. A man with a 30-year history of pulmonary sarcoidosis, confirmed by lymph node biopsy and a positive Kveim reaction, developed an incomplete bilateral bundle branch block pattern. Cardiac catherization and angiography revealed normal pressures, hemodynamic parameters and left ventricular contractile pattern; the coronary arteries were patent. His bundle electrograms demonstrated a normal H-V interval that was not altered by atrial pacing. After four weeks of steroid therapy the electrocardiographic abnormality disappeared. However, when restudied at this time, incomplete bilateral bundle branch block pattern could be elicited on atrial pacing and with the extrastimulus technique. Other electrophysiological observations suggested the presence of an AV nodal bypass tract and demonstrated changing intraventricular conduction patterns. The implications of these findings are discussed.


Sequential hemodynamic studies following mitral fascia lata valve replacement. Sequential hemodynamic studies were performed at mean intervals of 6 and 38 months following mitral valve replacement in 10 patients who had angiographically competent fascia lata valves and in whom preoperative hemodynamic studies were available. The cardiac index did not change significantly during postoperative studies as compared with the preoperative values, and its response in relation to oxygen uptake during exercise remained impaired. The mean pulmonary wedge and pulmonary artery pressures showed significant reduction at the first and the second postoperative studies, both at rest and during exercise. Progressive reduction in the pulmonary vascular resistance occurred in patients with high initial values, although the group mean did not alter significantly. The mean diastolic gradient across the mitral fascial valves averaged 8.1 +/- 1.3 mm Hg at rest rising to 22.5 +/- 2.4 mm Hg during exercise (P less than 0.001), and the calculated mitral valve area was 1.9 +/- 0.22 cm2 at rest and 2.36 +/- 0.3 cm2 during exercise (P less than 0.02). This study indicates that the insertion of stented mitral fascia lata valves results in significant hemodynamic improvement, which is maintained at least for 3 years after surgery, in patients with competent valves. However, a degree of obstruction to the forward flow is apparent especially during exercise, despite the central flow design of the valve.


Hemodynamic effects of lidoflazine during graded levels of bicycle exercise in normal subjects. The hemodynamic effects of lidoflazine were studied in 12 young healthy subjects who received the drug orally (240 mg daily) for 8 weeks. During exercise after lidoflazine, heart rate (-2.6%), mean arterial pressure (-3.1%), arterio-venous oxygen (A-V02) difference (-3.4%), pressure rate product (-6.2%), and systemic vascular resistance (-8.6%) were significantly lower, while cardiac output (+5.4%) and stroke volume (+8.3%) were significantly greater. The maximal heart rate was lower after lidoflazine (-6 beats/min) but the maximal oxygen intake (VO2max) was not affected by the drug. The major hemodynamic effects of lidoflazine appear to be, on the one hand, an unexplained decrease in heart rate, and on the other, a decrease in systemic vascular resistance; the latter, which was expected from a vasodilator, could account for the greater stroke volume and contributes to the decrease of the pressure rate product. Lidoflazine has another unexplained effect, i.e. a decrease of the A-VO2 difference, suggesting a decreased peripheral extraction of oxygen at rest as well as during submaximal and maximal exercise.


Assessment of immediate prognosis in acute myocardial infarction by a new noninvasive hemodynamic index. A prognostic index for acute myocardial infarction was developed from noninvasively accessible parameters, gathered prospectively within 24 h from the onset of symptoms in 185 consecutive patients. Of the 35 patients who died in hospital, 30 had power failure. The items subjected to discriminant function analysis were: sex, age, number of previous infarctions, present infarction transor nontransmural, heart rate, systolic arterial pressure (SAP), left ventricular ejection time (LVET), cardiothoracic ratio, and grade of pulmonary venous congestion scored 0-3 (PVC0-3). The items possessing the best predictive power were, in the order of their strength: age, SAP, LVET and PVCO-3; i.e., hemodynamically, afterload, stroke volume and preload. The discriminant function (DF) giving the prognostic score was: DF=3.9Xage(yr)-1.3X SAP (mm Hg) - 1.4 X LVET (msec) + 25.3 X PVC0-3 + 775.3 Score 550 was exceeded by 87% of the patients dying of power failure and only 16% of the survivors, and it was less in 84% of the survivors and 13% of those dying. To test the validity of the index it was applied to another series consisting of 100 consecutive patients and very similar results were obtained, suggesting that the index is of practical value in predicting hemodynamic deterioration early and by simple noninvasive means.


Transcutaneous aortovelography: reproducibility in adults and children. Transcutaneous aortovelography (TAV), a new ultrasonic technique for measuring instantaneous mainstream blood velocity in the aorta, is described. The technique uses the Doppler shift principle. The highest local blood velocity within the aortic arch can be recorded from a transducer placed in the suprasternal notch. Inspection of the systolic complexes recorded allows measurement of peak velocity, systolic complex area (proportional to ejected stroke volume), mean (time averaged) blood velocity and initial systolic acceleration. Formal analysis of variance was performed on measurements made by the five observers on 11 healthy adults and by 2 observers on 10 healthy children. Coefficients of variation of from 4 to 8% were found of all variables other than acceleration where the variation was up to 16%. Peak velocities in adults ranged from 97 to 130 cm/sec and were higher in children.


Radiation pericarditis and myocardial fibrosis. The case of a 45-yr-old man with constrictive pericarditis due to radiation for Hodgkin's disease is described. After pericardiectomy and clinical condition did not improve. At necropsy an extensive fibrosis of the myocardium especially located in the anterior part of the heart was found. The clinical consequences of this finding with respect to surgical treatment are briefly discussed.


Dependence of calcium release, tension generation and restoring forces on sarcomere length in skinned cardiac cells. The ascending limb of the length-tension diagram was studied in single skinned (sarcolemma-free) cardiac cells of rat ventricle. The free (Ca2+) in the perfusing solutions was buffered with ethyleneglycol-bis(beta-aminoethyl)N,N'-tetraacetic acid (EGTA). Tension was recorded with a photo-diode force transducer (detection limit: 1 mug, compliance 2-3 mum/mg) and was expressed as a function of the sarcomere length (SL) measured during contraction with a high-speed movie camera. When the SL was decreased by a brief (1-2 sec) exposure to  high free (Ca2+), a restoring force qas observed upon return of the cell to a relaxing solution. The restoring force was comprised of two components: (1) a rapid elongation to 1.57 identical to 0.06 mum developing a negative tension (relaxing force) as large as 4% of the maximum positive tension, which was observed in both skinned cells and in single myofibrils, and (2) a slow elongation from 1.57 identical to 0.06 to 1.93 identical to 0.14 mum, which was observed in skinned cells but not in single myofibrils. When the SL was kept extremely short by continuous Ca2+ activation for more than 30 sec, a shortening of the A band much below 1.0 mum persisted for several minutes after imposed re-elongation of the sarcomers (delta state). The amplitude of the tonic tension developed by direct activation of the myofilaments in the presence of high total (EGTA) was maximum for 2.20 mum SL and decreased very little when the SL was decreased. However, the decrease of tension became more pronounced at SL shorter than 1.55 mum when polyvinylpyrrolidone was added to the solutions at a concentration reducing the swelling of the myofilament lattice. This finding imposes some caution in applying the results obtained in skinned cardiac cells to intact tissue. The SL-tension diagram of the phasic contractions induced by Ca2+-triggered of Ca2+ in the presence of a slight EGTA buffering was similar to the SL-tension diagram of the intact rat ventricular muscle when the latter was expressed as a function of the active SL. In contrast, the SL-tension diagram of caffeine-induced phasic contractions was similar to that of the tonic tension produced by direct activation of the myofilaments. Decreasing SL results therefore in a partial inhibition of the Ca2+ triggered release process. It was concluded that Starling's Law may correspond to a length-dependence of several mechanisms including Ca2+-triggered release from the SR, interaction between thick and thin filaments and restoring forces.


Postextrasystolic potentiation in man.

Postextrasystolic potentiation in man. Postextrasystolic potentiation (PESP) is a pre- and afterload-independent property of the myocardium. Therefore it has been used to study left ventricular (LV) contractile state in patients with coronary heart disease and normals. Using a computer-controlled cardiac stimulator, regular rhythms with just above sinus frequency were applied to the right atrium and interrupted after every 20th beat by one shorter interval after varying delays. The next stimulus interval was corrected in such a way that the resulting R-R interval was the same as the basic R-R interval. Control of the postextrasystolic R-R interval is a prerequisite for meaningful and quantitative evaluation of PESP. Of 166 basic and postextrasystolic beats in 12 patients LV pressures (P), obtained with a catheter tipmanometer, were analyzed. There was no significant difference in enddiastolic P between pre- and postextrasystolic beats. Peak dP/dt, time to peak dP/dt, (dP/dt)/P at a left ventricular P of 50 mm Hg and Vmax were used as indices of left ventricular contractile state. It was found that on increasing prematurity of the extrasystolic beat, there was a progressive and reproducible increase of these indices (selfevidently a decrease in the case of the time to reach peak dP/dt). Using Vmax as an index of contractile state, no significant PESP could be demonstrated in the two patients with very enlarged left ventricular volumes and depressed ejection fractions. PESP, elicted in this (only allowable) way, is an integral, quantitative and reproducible method for LV contractility estimation in man.


Calibrated apexcardiography and assessment of left ventricular dynamics in man. Using a calibrated displacement transducer, the total amplitude of the systolic wave of the left apexcardiogram (S), its first derivative (dS) and the normalized first derivative (nS), were evaluated as noninvasive indices of left ventricular function in human subjects. A strong correlation was present between peak dS and S in normal subjects (r=0.95, P less than 0.001). At an identical S, abnormal hearts had a lower peak dS, and this allowed a separation between groups with normal and abnormal ventricular dynamics. The index peak nS was significantly lower in patients with congestive cardiomyopathy (P less than 0.005) and ischemica heart disease in the presence of a low ejection fraction (P less than 0.001). It correlated significantly with LVEDP (r=-0.69, P less than 0.001), with ejection fraction (r=0.66, P less than 0.001) and with left ventricular contractility indices derived from isovolumic left ventricular pressure and its first derivative, recorded simultaneously by means of high fidelity micromanometers (peak VCE: r=0.76; Vmax total pressure: r=0.70; peak dP/dt: r = 0.69; P less than 0.001). The index peak nS was superior to S and peak dS, being less variable, independent of thorax circumference and better correlated with hemodynamic parameters. A close relationship was also present between the total amplitude of the atrial wave (A) and its peak first derivative (peak dA) in normal subjects (r = 0.98, P less than 0.001). For an identical A wave amplitude, patients with an increased left ventricular anddiastolic volume had lower values for peak dA (P less than 0.001).


The analysis of size, shape and contraction pattern of the right ventricle from angiocardiograms. This paper describes an approach to assess quantitatively videoangiocardiograms of the geometrically complex right ventricle with the aid of an automated roentgen-television-computer system. The influence of spatial positions and heart phase on the accuracy of right ventricular volume determination with biplane and single plane area-length and multiple slices methods was defined on the basis of a special cast study. Without considering position and phase a relative deviation - with a biplane procedure - of not equal to 11.9% has to be faced, which will be reduced to not equal to 8.8% when position, and to not equal to 6.2% when in addition the phase of the cardiac cycle is taken into consideration. Fourier analysis was applied to biplane silhouettes and 'true' cross-sections of mechanically sliced human right ventricular casts in order to arrive at 3-D 'norm ventricles' as reference for size and shape analysis of beating normal and diseased hearts. For contraction pattern analysis the dimensional information from each X-ray plane was processed independently. Selected points (anatomical landmarks), being marked with an x/y coder, served as internal reference system. Multiple parallel diameters, their directions given by a pair of indicated points or by a freely selected angle, were followed throughout the cardiac cycle. Applied was the method to angiocardiograms with normal hearts and those with ASD, pulmonic stenoses, and postoperative tetralogies of Fallot. It can be concluded that on the basis of videoangiocardiograms (1) right ventricular volume can be determinded with a defined and acceptable accuracy, with both biplane and single plane procedures, if one takes into consideration the spatial position and the cardiac phase, (2) right ventricular shape can be quantitated for an objective description, and (3) right ventricular contraction and relaxation processes can be assessed and quantitated for normal and diseased right ventricles.


Vertebrate striated muscle: length dependence of calcium release during contraction. It is well known that the force-generating ability of vertebrate striated muscle depends upon its length. At lengths longer than the optimum there is a close correlation between decreasing overlap of the myofilaments and the lessening of contractile force, but at lengths shorter than the optimem additional factors may contribute to decreased contracility. For example, the release of calcium into the myofilament space is decreased during contractions at short muscle lengths, and the morphological changes seen in light and electron micrographs of shortening fibers suggest that the decreased calcium release occurs in the core of the fiber. This length dependent change in calcium release may be one of the factors determining the decrease in contractility with shortening.