Rabbit ovarian protein kinases. III. Gonadotrophin-induced activation of soluble adenosine 3',5'-monophosphate-dependent protein kinases. Cytosol of mature estrous rabbit follicles contains a single species of protein kinase, protein kinase 3, which can be classified as a type II cAMP-dependent protein kinase. Cytosol of functional rabbit corpora lutea (CL) contains, in addition to protein kinase 3, a second species of kinase activity, protein kinase 2, which can be classified as a type I cAMP-dependent protein kinase. These conclusions are based upon the relative dissociation and reassociation characteristics of the two holoenzymes in the presence and absence of 0.5 M NaCl after in vitro dissociation by cAMP, upon the effect of MgATP on salt- and basic protein-induced dissociation, and upon their relative elution from DEAE-cellulose. Protein kinase 3 in mature estrous rabbit follicles was rapidly activated after an iv injection of hCG. The activation was demonstrated by an increase of the protein kinase activity ratio as well as by the appearance of the free catalytic subunit of protein kinase upon Sephadex gel filtration. Maximal activation occurred within 10 min of in vivo hormone administration and required ovulatory doses of hormones with LH-like activity. Neither PRL, ACTH, epinephrine, nor a highly purified preparation of FSH promoted activation of the follicular protein kinase 3. Demonstration of protein kinase activation in follicles was achieved in the presence of 0.5 M NaCl in the homogenization media. After an iv injection of hCG, a partial activation of luteal protein kinases 2 and 3 was demonstrated, as reflected by the increase of the protein kinase activity ratio. These results implicate an important role for cAMP-dependent protein kinase 3 in LH action in rabbit ovarian follicles and for cAMP-dependent protein kinases 2 and 3 in LH action in rabbit CL.


Converting enzyme inhibition augments and competitive inhibition of angiotensin II partially restores reflex adrenal catecholamine release in anephric dogs. Adrenal epinephrine (E) release after hemorrhage in anesthetized dogs is blunted by acute nephrectomy and restored by angiotensin II infusion. In the present study, we report the effect of converting enzyme inhibition by SQ 20881, a decapeptide, and of competition inhibition of angiotensin II by saralasin (1-Sar-8-Ala-Ang-II) on reflexly stimulated adrenal release of E and norepinephrine (NE) in three groups of acutely anephric dogs. Aortic catheters and adrenal vein to femoral vein Silastic shunts were placed in dogs anesthetized with pentobarbital and mechanically ventilated. Adrenal secretion rates were calculated from adrenal vein to aorta catecholamine concentration differences divided by measured adrenal venous flow. Catecholamine concentrations were determined with trihydroxyindole technique. Blood samples were obtained before and 15, 30, and 60 min after rapid hemorrhage to a stable mean arterial pressure of 50 mm Hg. Saralasin infusion (10 microgram/kg/min) supported adrenal E release in anephric hemorrhaged dogs toward secretion rates comparable to those seen in intact dogs. Anephric SQ 20881 (approximately 0.5 microgram/kg) recipients had delayed (60 min) augmented adrenal E and NE release after hemorrhage. In resting animals not reflexly stimulated by hypovolemia, neither drug provoked adrenal E or NE release. These results suggest an agonist effect of saralasin on reflex adrenal E release and increased responsiveness of the stimulated adrenal medulla under the influence of converting enzyme inhibition.


Studies on the dissociation of prolactin from mammary tumor membranes. The dissociation of [125I]iodo-PRL from crude membrane preparations of two transplantable rat mammary carcinomas was studied. At least 1/2 of the specifically bound PRL remains undissociated when membrane pellets are resuspended in buffer. The apparent irreversibly bound fraction can be removed at least partially upon incubation with 6 M urea, but not with 1 M urea. By all the criteria used, the dissociated PRL was undamaged. The physiological importance of this phenomenon may relate to the presence of more than one type of binding site which current techniques do not allow us to distinguish.


Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary. The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.


Interactions of gonadotropins with corpus luteum membranes. II. The identification of two distinct surface membrane fractions from superovulated rat ovaries. Fractions enriched in hCG-binding activity were prepared by differential rate centrifugation of superovulated rat ovarian homogenates and were applied to continuous sucrose density gradients (20-55%). After centrifugation at 63,000 x gav for 3.5 h, fractions of each gradient were collected and assayed for a range of marker enzyme activities characteristic of surface membranes and subcellular organelles. Mitochondria, lysosomes, and rough and smooth endoplasmic reticulum membranes accumulated in the gradient between 38-41% sucrose (1.165-1.180 g/cm3). Nuclei passed through the gradient. However, the various surface membrane markers concentrated in two distinct regions of the gradient. Alkaline phosphatase, phosphodiesterase, (Na+ + K+)ATPase I, and hCG-binding activity concentrated at 29-32% sucrose (1.120-1.135 g/cm3), whereas 5'-nucleotidase, Mg2+-dependent ATPase, and adenylate cyclase activities (and minor peaks of hCG-binding and phosphodiesterase activities) were enriched at 36-38% sucrose (1.16-1.17 g/cm3). A second ATPase, [(Na+ + K+)ATPase II], was also observed in this region of the gradient, which could be distinguished from (Na+ + K+)ATPase I of the light membrane fraction by its sensitivity to the Ca2+-chelating agent, ethylene glycol bis-(aminoethyl)tetraacetic acid (EGTA). The kinetics of binding of radioiodinated hCG to the gonadotropin receptors of the light and heavy membrane fractions were very similar. It is suggested that fractionation of superovulated rat ovaries yields two distinct populations of surface membrane material which have distinct densities and marker enzyme profiles. Furthermore, in contrast to the heavy membrane fraction, light membranes seem to possess considerable amounts of hCG receptor activity but very little adenylate cyclase.


alpha-Adrenergic inhibition of adenosine 3',5'-monophosphate accumulation and parathyroid hormone release from dispersed bovine parathyroid cells. The possibility of alpha-adrenergic modulation of cAMP accumulation and parathyroid hormone (PTH) release was investigated in dispersed bovine parathyroid cells. cAMP accumulation due to the mixed alpha- and beta-adrenergic agonists, (-)epinephrine and (-)norepinephrine, was significantly enhanced by the alpha-adrenergic inhibitor phentolamine; that due to the "pure" beta-adrenergic agonist, (-)isoproterenol, was not altered significantly. Direct inhibition of agonist-stimulated cAMP accumulation was effected by adding increasing concentrations of (-)epinephrine to concentrations of (-)isoproterenol maximally stimulating cAMP accumulation. A 50-75% inhibition of cAMP was observed which was specifically blocked by phentolamine. This inhibition was not specific for beta-adrenergic stimulation, as (-)epinephrine also inhibited dopamine-stimulated cAMP accumulation. The inhibition of (-)isoproterenol-stimulated cAMP accumulation by (-)epinephrine was unaffected by ambient calcium concentration. Stimulation of PTH release by (-)epinephrine and (-)norepinephrine was potentiated by phentolamine and inhibited by the beta-adrenergic blocker, (-)propranolol, demonstrating alpha-adrenergic modulation of hormone release and confirming the close relationship between cAMP accumulation and PTH release previously shown in this system. These results demonstrate the presence of an alpha-adrenergic receptor in dispersed bovine parathyroid cells which inhibits agonist-stimulated cAMP accumulation and PTH release by a mechanism independent of extracellular calcium.


Studies on the site of action of vasopressin in inducing adrenocorticotropin secretion. Hypophysectomized rats bearing three transplanted pituitaries under the kidney capsule responded to synthetic lysine vasopressin or pitressin with a significant elevation of plasma corticosterone, whereas hypophysectomized rats with no grafts did not. This response was completely abolished by pretreatment of animals with dexamethasone but was unaltered by central hypothalamic destruction. Corticotropin-releasing factor content of the hypothalamic median eminence, hypophyseal stal-, or pars nervosa of the posterior pituitary of intact rats was unchanged 5 or 10 min after ip injection of vasopressin compared to the basal level. We conclude that vasopressin and dexamethasone act directly on the adenohypophysis in vivo to exert their stimulatory or inhibitory effect on ACTH secretion.


Organization of the medial hypothalamus for control of adrenocorticotropin in the cat. To examine the role and interrelations of areas of the medial hypothalamus in the control of release of ACTH, we stimulated electrically (20-sec train, 200-microamperemeter amplitude at 100 Hz) 695 sites in the hypothalamus of 91 cats anesthetized with chloralose-urethane. Changes in ACTH were measured by RIA. Responses of arterial pressure could not account for changes of release of ACTH. Several ACTH-active areas were defined. The anatomical relations of these areas with known nuclei and pathways then were considered. Two ACTH facilitatory areas and one ACTH inhibitory area were identified in the lateral aspect of the medial hypothalamus. The dorsal facilitatory area appears to be an extension of the lateral division of the dorsolongitudinal fasciculus and to extend medially to join the Fields of Forel, the ventral tegmental area of Tsai, and the parvocellular, paraventricular, and periventricular nuclei. The ACTH inhibitory area appears to be an extension of portions of the central tegmental tract and to extend medially to the posterior hypothalamic area and the dorsal hypothalamic area and ventrally toward the basal hypothalamus. The ventral ACTH facilitatory area appears to be coincident with the medial forebrain bundle and to extend anteroventrally and medially through the supraoptic decussation to the suprachiasmatic, ventromedial, dorsomedial, periventricular, infundibular, and premammillary nuclei. Stimulation of the median eminence led to increased release of ACTH. The results suggest that ascending pathways from the lower brainstem mediating control of ACTH project to discrete areas of the hypothalamus and then converge on the medial basal hypothalamus.


The response of adenosine 3',5'-monophosphate to norepinephrine in the hypothalamus and pineal organ of female rats in proestrus or diestrus. The response of cAMP to norepinephrine (NE) was measured in hypothalamic tissue and pineal glands of cycling female rats killed on the mornings of proestrus and diestrus (in 4-day cycles) or diestrus II (in 5-day cycles). The results from 4- or 5-day cycles were similar in all cases. The report of Weiss and Crayton that the response of adenylate cyclase in pineal homogenates to NE is reduced on proestrus was confirmed, and a similar but lesser reduction was also detected with intact pineal glands incubated in vitro. Chopped hypothalamic tissue incubated in vitro revealed a greater response of cAMP levels to NE on proestrus compared to diestrus in the anterior hypothalamic-preoptic area, but no significant changes in the middle or posterior hypothalamus. In an effort to reproduce the changes noted on proestrus, ovariectomized animals were injected with estradiol benzoate for 2 days, but no effects of the treatment on the cAMP response to NE were found. If the animals were also given reserpine, however, the estrogen was effective in reducing the response of pineal adenylate cyclase to NE. Possible explanations for these results and the potential significance of alterations in NE receptor systems for the control of gonadotropin release are discussed.


Alterations in insulin binding induced by changes in vivo in the levels of glucocorticoids and growth hormone. Previous studies have shown that the sensitivity of tissues to insulin is diminished in states of glucocorticoid and GH excess and is increased when these hormones are deficient. To evaluate the role of the insulin receptor in these states, we have studied [125I]insulin binding to purified liver plasma membranes obtained from rats with a variety of perturbations of both glucocorticoids and GH. Glucocorticoid excess was produced in rats by administration of ACTH (40 U/day for 4 days) or dexamethasone (1 mg/day for 4 days). This resulted in an insulin-resistant state. Associated with this insulin resistance, there was a 50-60% decrease in insulin binding to its specific receptors in liver. Conversely, adrenalectomy, which produces an increase in insulin sensitivity, was associated with an increase in insulin binding to liver. Computer-assisted Scatchard analysis using a negative cooperative model for the inulin-receptor interaction indicated that, in contrast to our findings with obesity, the changes in insulin binding in these states were most likely due entirely to changes in receptor affinity, with no change in receptor concentration. GH administration also produced mild insulin resistance and a decrease in receptor concentration. This was associated with a reciprocal increase in receptor affinity and thus, no major alteration in insulin binding occurred at low physiological insulin concentrations. Hypophysectomized rats, on the other hand, showed an increase in receptor concentration and a decrease in affinity, and GH treatment only partially corrected these changes. Rats implanted with the MtT tumor (which secretes ACTH, GH, and PRL) have the combined effects of excess glucocorticoids and GH and are very insulin resistant. Liver membranes prepared from these rats showed a decrease in insulin binding and receptor affinity similar to that observed in other states of glucocorticoid excess. Further, adrenalectomy of the tumor-bearing rats resulted in an increase in insulin binding despite the persistence of the elevated levels of GH, ACTH, and PRL. These findings suggest that alterations in insulin receptor affinity and number may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess and deficiency, and follow hypophysectomy. In contrast, the insulin resistance associated with GH excess is mediated at either a site on the receptor distal to the insulin-binding site (i.e. transduction) or at one or more of the intracellular reactions important in insulin action.


Rat adrenal androgen receptor: a possible mediator of androgen-induced decreased in rat adrenal weight. Many previous studies have demonstrated effects of gonadal steroids on adrenal weight in the rat. Most of these effects are indirect, depending upon alterations in the pituitary-adrenal axis for their expression. In this study we have attempted to examine the direct effects of gonadal steroids on adrenal weight in the rat. This was done using hypophysectomized, castrated male rats receiving ACTH replacement, a model which excludes pituitary-adrenal feedback effects. Estradiol-treated rats did not differ from controls, whereas testosterone-treated rats exhibited a small but statistically significant decrease in adrenal weight. As a first step in exploring the mechanism of this androgen effect, we have identified a specific dihydrotestosterone-binding protein in the rat adrenal gland. A single class of high affinity (Kd = 0.6-2.0 x 10(-8) M), saturable (28 fmol/mg cytosol protein), cytoplasmic binding sites was found using both protamine sulfate precipitation and dextran-coated charcoal assays. The specificity, sedimentation coefficient on sucrose gradient, and sensitivity to sulfhydryl reagents and heat of this dihydrotestosterone-binding protein are typical of the cytoplasmic androgen receptor from other androgen target tissues such as prostrate. We conclude that testosterone can decrease rat adrenal weight directly, and that the mechanism may involve a high affinity binding protein, as has been shown in other androgen-responsive systems.


Evidence for activation by beta2-adrenergic receptors of adenosine 3',5'-monophosphate formation in Ehrlich ascites tumor cells. The existence of aminergic receptors in mouse Ehrlich ascites tumor cells was studied. L-Isoproterenol in vitro stimulated the formation of cAMP in isolated Ehrlich ascites tumor cells. Stimulation by isoproterenol of cAMP formation was not significantly inhibited by practolol, a beta1-adrenoceptor antagonist-Salbutamol, a beta2-adrenoceptor agonist, markedly stimulated the formation of cAMP in Ehrlich ascites tumor cells at concentrations from 10(-8)-10(-3) M. After the addition of salbutamol, cAMP levels reached a maximum in 10 min and declined to about 2-fold of the basal level to 30 min. The stimulation by salbutamol of cAMP formation was markedly inhibited by butoxamine, a beta2-adrenoceptor antagonist, but not by practolol. Furthermore, the effect of a maximal dose of salbutamol was additive to that of prostaglandin E2. Histamine and 4-methylhistamine, a histamine H2 receptor agonist, had no significant effects. Therefore, it is suggested that a beta2-adrenergic receptor exists in the membranes of Ehrlich ascites tumor cells in terms of the adenylate cyclase-cAMP system.


Progressive suppression of adrenocorticotropin secretion in resting adrenalectomized dogs by low stepwise infusions of cortisol. Ten male mongrel dogs of 8.3-14.3 kg were bilaterally adrenalectomized and maintained on replacement steroids for 1 week. No exogenous steroids were detectable at the time of the experiment. Each dog, under light Nembutal anesthesia, received two stepwise primed constant infusions of cortisol (iv), each lasting 90 min (except in one dog with three infusions of 60 min). A recovery period of 80 min without cortisol infusion followed. Twenty-three venous blood samples were withdrawn for determination of plasma ACTH by adrenal cell suspension bioassay and 25 were withdrawn for plasma cortisol by fluorimetry at various times before, during, and after the cortisol infusions. ACTH secretion rates were calculated continuously from functions fitted to ACTH concentration and time data, using a validated single compartment approach with previously determined clearances and volumes. Initial high ACTH secretion rates of 1.0 +/- 0.2 mU/kg . min (range, 0.3-2.4) were reduced by 67.4 +/- 4.6% and 94.6 +/- 2.1% by the two cortisol infusions, which yielded cortisol plateau concentrations in plasma of 4.24 +/- 0.41 and 6.09 +/- 0.68 microgram/100 ml, respectively. This sensitive feedback response to low cortisol concentrations was delayed, with no effect for about 20 min and maximal effect reached within 1 h. There was no evidence of a fast rate-sensitive feedback. Some increases in ACTH secretion were seen during the recovery period (errors are +/- SEM).


Specific binding of iodinated growth hormone to rat liver in vivo. The specific uptake by rat liver of human (hGH) and bovine (bGH) GHs labeled with 125I was studied by an in vivo procedure. A significant reduction of the uptake was observed when labeled hormones were injected together with different amounts of the corresponding native GH. This reduction was dose dependent, and the concentration of native hormone that prevents 50% of the liver uptake of the labeled hormone was close to 12 microgram/100 g BW. In normal rats, only native hGH or bGH significantly decreased the liver uptake of [125I]iodo-bGH, while bovine PRL (oPRL) or heat-denatured bGH were inactive. The highest inhibition of the uptake of [125I]iodo-hGH by rat liver was obtained when this labeled hormone was injected either together with hGH or with bGH plus oPRL while partial displacement was observed with bGH or oPRL. These data suggest that hGH binds to both somatotropic and lactogenic sites in the liver of normal rats. In hypophysectomized animals, only the somatogenic binding sites could be detected.


The contribution of local tissue thyroxine monodeiodination to the nuclear 3,5,3'-triiodothyronine in pituitary, liver, and kidney of euthyroid rats. The contributions of local T4 monodeiodination and plasma T3 to the nuclear T3 of anterior pituitary, liver, and kidney were measured in euthyroid rats. After injection of [125I]T4, there was a gradual increase in the quantity of plasma [125I]T3 in excess of injected contaminant, which peaked at approximately 12 h after injection and remained a constant fraction of plasma [125I]T4 (2.8 X 10(-3) after that time. In the nuclei of anterior pituitary tissue, there was also a slow increase in locally produced [125I]T3 (in excess of that which could be accounted for by plasma [125I]T3) which appeared to peak at about 16 h after [125I]T4 administration. The ratio of nuclear [125I]T3 generated intracellularly to plasma [125I]T4 was constant at 18 and 24 h after T4 injection and was 13 +/- 2 X 10(-3) in nuclei of pituitary, 2.0 +/- 0.4 x 10(-3) in liver, and 0.47 +/- 0.1 x 10(-3) in kidney (all values are mean +/- SD). This locally generated T3 resulted in a markedly higher nuclear to plasma (N:P) ratio for [125I]T3 than for injected [131I]T3 in the same animals. The N:P ratio for [125I]T3 at equilibrium after injected T4 was 2.4 +/- 0.6, 0.47 +/- 0.09, and 0.10 +/- 0.03 (nanograms of T3 (mg DNA)-1/ng T3 ml-1) in pituitary, liver, and kidney. Comparable values for [131I]T3 N:P ratios were 0.47 +/- 0.14 (pituitary), 0.18 +/- 0.01 (liver), and 0.036 +/- 0.008 (kidney). Using RIA values for plasma T4 and T3 concentrations in these rats and maximal nuclear T3-binding capacities estimated in parallel experiments, the gravimetric quantities of nuclear T3 derived from plasma T3 and from local T4 to T3 monodeiodination were estimated and expressed as the percentage of saturation of T3 receptors. Seventy-eight percent of nuclear T3 receptor sites in anterior pituitary were occupied with one-half of the nuclear T3 derived directly from plasma T3 and the other half from intrapituitary T4 monodeiodination. Local T4 monodeiodination provided only 28% and 14%, respectively, of the nuclear T3 in liver and kidney, and the nuclear receptors of these tissues were about 50% saturated. Since our previous studies have shown that the occupancy of the pituitary nuclear T3 receptors may regulate TSH release, these data provide a mechanism by which TSH secretion could be altered by changes in either plasma T3 or T4, whereas nuclear T3 in liver and kidney is predominantly a function of the plasma T3 concentration.


16-Hydroxylation of estrone-3-sulfate and estrone in the guinea pig in vivo. [6,7-3H]Estrone-3-sulfate or [6,7-3H]-estrone of high specific activity was injected into adult female English Shorthair guinea pigs. Blood, liver, kidney, gall bladder bile, and urine were obtained and investigated for metabolites. Chromatographic procedures followed by enzymatic or solvolytic cleavage of conjugates and subsequent crystallization with appropriate carrier steroids revealed the pattern of metabolites formed. Injected estrone sulfate was partially hydrolyzed and reconjugated, resulting in the production of estrone and estradiol glucuronides. The main metabolites, however, were monosulfates of 16alpha-hydroxyestrone, 16-keto-17beta-estradiol, and estriol as well as disulfates of 16alpha-hydroxyestrone, estriol, and 16beta-hydroxyestrone. Particularly high amounts of these were found in urine. By far the main metabolites of injected estrone were glucuronides of estrone and estradiol, although the pattern of mono- and disulfated steroids was qualitatively similar to that found after estrone sulfate injection. It is concluded that the guinea pigs employed in the study hydroxylated estrogen in the 16alpha- and 16beta-configurations and that this activity was much more pronounced after injection of estrone sulfate than after estrone.


Studies on cyclic nucleotides in the adrenal gland. VIII. Effects of angiotensin on adenosine 3',5'-monophosphate and steroidogenesis in the adrenal cortex. Effects of angiotensin II on corticoid biogenesis and cAMP levels in the zona fasciculata-reticularis (the decapsulated fraction) and the zona glomerulosa (the capsular fraction) from the rat adrenal gland have been studied. Angiotensin II exclusively stimulated steroidogenesis in the zona glomerulosa without stimulation of the cAMP system, suggesting that steroidogenic action of this polypeptide does not involve the adenylate cyclase system. Angiotensin II was also found to stimulate cAMP-phosphodiesterase activity in the zona glomerulosa. An elevation of calcium concentration in the incubation medium has been observed to be effective in stimulating the production of aldosterone and cAMP by the capsular fraction. Angiotensin II caused a significant enhancement of the steroidogenic response of the capsular fraction to increasing calcium concentration regardless of the response of the cAMP system to calcium. This steroidogenic effect of angiotensin II was completely abolished by calcium antagonists added to the incubation medium without any inhibitory effect on the calcium-induced accumulation of tissue cAMP. These results suggest that angiotensin II acts on the adrenal II acts on the adrenal glomerulosa cell to increase intracellular calcium, which in turn directly stimulates steroidogenesis concomitant with the increased activity of phosphodiesterase.


Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma. Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.


Specificity of action of neuraminidase, according to its bacteriological origin. Neuraminidase from Vibrio Cholerae selectively cleaves sialic acid from FSH, but leaves the LH sialic acid, not influencing the biological activity of the latter hormone. On the other hand, Neuraminidase from Clostridium perfringens does not possess this specific action and destroys the biological activity of LH as is suggested by Parlow's OAAD-test.


Antiepileptic drugs and vitamin D supplementation. The incidence of biochemical signs of vitamin D deficiency and the effects of vitamin D supplementation were investigated in 83 children and 95 adults on chronic antiepileptic therapy and 40 mentally retarded controls living under comparable conditions. Low 25-hydroxyvitamin D and serum calcium, and elevated immunoreactive parathyroid hormone and alkaline phosphatase was a common finding in all groups, but in patients on antiepileptic drugs, signs of vitamin D deficiency were recorded more frequently. Supplementation of 125 microgram or 250 microgram vitamin D3 per week for 9 months normalized the laboratory findings in most patients; the effect of 37.5 microgram/week only slightly exceeded the influences of season observed in the controls and in epileptic patients without vitamin D. It is suggested that a dose between 37.5 and 125 microgram vitamin D3/week might be most suitable to avoid biochemical signs of vitamin D deficiency in children and adults on antiepileptic drugs.


Translation of alfalfa-mosaic-virus RNA 1 in the mRNA-dependent translation system from rabbit reticulocyte lysates. Translation of alfalfa mosaic virus (AMV) RNAs in the mRNA-dependent rabbit reticulocyte cell-free system was examined using different RNA concentrations. The pattern of products synthesized under the direction of AMV RNA 2, 3 and 4 was not or almost not influenced by their concentration. However, depending on the RNA 1 concentration either a very large protein of Mr 115,000 or a mixture of two smaller proteins, Mr 58,000 and 62,000 respectively, was formed. These three proteins represent overlapping peptide chains with identical N-termini. Addition of the cap analogue 7-methylguanosine 5'-monophosphate (m7GMP) or AMV RNA 3 stimulated the production of the 115,000-Mr protein at the expense of the 58,000-Mr and 62,000-Mr proteins. Both m7GMP and RNA 3 probably reduce the active concentration of RNA 1 by competing for (a) cellular component(s) necessary for translation. These experimental results suggest that the rate of translation beyond the C termini of the 58,000-Mr and 62,000-Mr proteins is reduced or completely inhibited owing to the limited availability of the succeeding tRNA(s).


Effects of spin-labeled stearates on duck erythrocyte adenylate cyclase. The effects of various spin-labeled stearates on duck erythrocyte adenylate cyclase were investigated. Only 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl caused an increase in adenylate cyclase activity. It increased the basal rate by about 50%, and the activities stimulated by isoproterenol and isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate by 35%. Upon analysis of the width parameter delta1 in the electron spin resonance spectra for both the basal enzyme activity and the stimulation obtained with effectors such as guanosine 5'-[beta,gamma-imido]triphosphate, isoproterenol, isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate and NaF, a correlation of the changes of modification in adenylate cyclase activities was found. These findings suggest that the molecular environment of the enzyme has been modified.


Parathyroid hormone and calcitonin levels in vitamin D deficient rickets. In six infants aged between 5 and 8 months with vitamin D deficient rickets, we have studied blood levels of calcium (Ca), phosphorus (P), alkaline phosphatase, immunoreactive parathyroid hormone (PTH) and calcitonin (CT), as well as urinary excretion of Ca, P, hydroxyproline and cyclic AMP, both under basal conditions and during a 4h infusion of 20 mg/kg 10% Ca gluconate in normal saline. Under basal conditions all the infants had high alkaline phosphatase (range: 470--770 U.I./1); PTH (range: 620--1200 pg Eq/ml) and CT (range: 440--750 pg/ml) but two infants had hypocalcaemia and four had normocalcaemia and hypophosphataemia. The urinary Ca excretion was low whereas the urinary P, hydroxyproline and cyclic AMP excretions were high. During Ca infusion the total serum Ca and CT levels increased, while alkaline phosphatase and PTH fell. After the end of the infusion, CT levels fell perceptibly; phosphaturia, hydroxyprolinuria and cyclic AMP decreased on the day of the infusion.


Prolactin-producing pituitary adenoma in a 9 year old boy. A boy aged 9 years and 8 months was evaluated for headache and an enlarged sella. His neurological status and visual fields were normal. After injection of radiographic contrast agent, computerized axial tomography showed evidence of an intrasellar tumor. The most striking endocrine abnormalities were growth hormone insufficiency after arginine infusion and after insulin-induced hypoglycemia, and excessively elevated prolactin levels ranging between 1220 ng/ml and 1560 ng/ml. A slightly granulated, acidophilic pituitary adenoma was selectively removed by the trans-sphenoidal approach. The function of the anterior pituitary improved post-operatively. Growth hormone secretion after insulin-induced hypoglycemia returned to normal, and the basal serum prolactin levels decreased, but are still three times higher than normal.


Humoral hypercalcaemia in a patient with renal-cell carcinoma. In a patient with hypercalcaemia secondary to a renal-cell carcinoma, a concentration gradient of bioactivity was detected between the tumour effluent vein and the peripheral venous blood that was capable of stimulating adenylate cyclase in bone cells. Immuno-reactive PTH was undetectable in the tumour effluent and in the peripheral blood. It is concluded that a non-parathyroid humoral factor whose action involved cyclic AMP stimulation was responsible for the hypercalcaemia.


Effect of polyenyl phosphatidyl choline on clofibrate-induced increase in LDL cholesterol. In a double-blind, randomised, cross-over trial clofibrate and a combination of polyenyl phosphatidyl choline (PPC) plus clofibrate were tested in 67 patients with hyperlipoproteinemia. Each treatment lasted for 4 weeks and was separated by a 4 week placebo period. The daily doses were clofibrate 1.2 g and PPC 1.8 g + clofibrate 1.2 g. respectively. The results revealed that polypenyl phosphatidyl choline prevented the elevation of LDL-cholesterol induced by clofibrate treatment, and that the lipid-lowering potency of the combination did not differ significantly from that of clofibrate. Since elevation of LDL-cholesterol is considered to increase the risk of coronary heart disease, the combination appears to offer a therapeutic advantage. Despite the significance of this clinical observation, a final decision may only be obtained from a prospective, long term investigation in patients with coronary heart diseases and hyperlipoproteinemia.


Effects of adenosine on neurally mediated norepinephrine release from the cat spleen. The effects of adenosine on the release of 3H-norepinephrine (3H-NE) from the isolated, perfused cat spleen consequent to nerve stimulation were evaluated. Electrical stimulation of the splenic nerve (5 Hz/100 impulses total) caused a release of 3H-NE and a rise in perfusion pressure. Adenosine added to the perfusion fluid (final concentrations 1 X 10(-6), 1 X 10(-5), and 1 X 10(-4) M) significantly reduced the pressure response elicited by nerve stimulation. In addition, adenosine (1 X 10(-4) M) slightly increased the release of total 3H consequent to nerve stimulation. Theophylline (1 X 10(-4) M) produced both a slight increase in the release of total 3H and a diminished pressure response. The effects of adenosine were effectively antagonized by this concentration of theophylline. Neither substance had any effect on the spontaneous release of total 3H. Adenosine (1 X 10(-4) M) also antagonized the pressure response elicited by perfusion of NE.


The natural tolerance of the afghan pika (Ochotona rufescens) to morphine. A lagomorph, the afghan pika, Ochotona rufescens showed no effect whatever following the subcutaneous injection of morphine in doses up to 50 mg per 100 g of body weight, i.e. 250 times the ED50 for the rat. Higher doses were toxic and induced convulsions. However, the pika is responsive to synthetic opiates such as etorphine, pentazocine and phenoperidine. Interestingly enough, morphine antagonized the opiate response elicited by those narcotics to which the animal is sensitive. Pharmacokinetic analysis demonstrated that morphine enters the pika's brain as readily as it does the rat's. In addition, opiate receptor sites, which are present in normal amounts in pika brain retained their high affinity for 3H-etorphine (KD = 0.3 nM), 3H-naloxone (KD = 1.2 nM) and morphine. Moreover, binding of morphine to pika brain homogenates was inhibited in the presence of sodium ions (agonist response). Therefore, the antagonism of phenoperidine action by morphine appeared not to occur at the opiate receptor site; the mechanism of the pika's natural tolerance to morphine may reside in molecular events that normally preceed (metabolism?) or follow (enzyme activation?) the interaction between the drug and its specific recognition sites.


Inhibition of nigral and neocortical cells by gamma-hydroxybutyrate: a microiontophoretic investigation. gamma-Hydroxybutyrate (GHB) and gamma-aminobutyrate (GABA) were applied microiontophoretically near spontaneously active cells in the substantia nigra and the neocortex of chloral hydrate-anaesthetized rats. Whereas GABA in "low doses" (i.e. 20 nA ejection currents) depressed the activity of cells in both areas, GBH reduced the firing rate only when the compound was expelled with two to three times higher ejection currents. Under these conditions GHB depressed about half of the nigral but the majority of the neocortical cells tested. The depressant action of GABA was readily antagonized by microiontophoretic bicuculline methiodide whereas the effects of GHB were resistant to this alkaloid. Intraperitoneally administered GHB in doses of 300 and 600 mg/kg reduced the firing rate of the majority of spontaneously active neocortical cells. In conclusion, GHB inhibits the firing of nigral and neocortical neurons. However its effects are not mediated through the activation of bicuculline-sensitive GABA receptors.


Mechanism of quasi-morphine withdrawal behaviour induced by methylxanthines. Of 7 phosphodiesterase inhibitors tested for ability to induce a quasi-morphine withdrawal syndrome (QMWS) in opiate-naive rats, five were effective in a dose-related way. These, in descending order of potency, were IBMX, ICI-63197, RO-201724, theophylline and caffeine. Their potencies in inducing a QMWS correlated significantly (P less than 0.05) with those in inhibiting low Km cyclic AMP phosphodiesterase of rat brain homogenate. There was no correlation with potencies in inhibiting cyclic GMP phosphodiesterase.


Effects of intravenous baclofen on dorsal horn neurons of spinal cats. Intravenous baclofen (1 mg/kg) abolished or severely attenuated the high threshold late component (but not the low threshold early component of long duration (greater than 150 msec) discharges evoked in cat dorsal horn neurons by intense transcutaneous electrical stimulation. Baclofen similarly reduced the spontaneous activities and discharges evoked by intra-arterial bradykinin in these same cells. These powerful inhibitions of dorsal horn interneurons may be the basis of baclofen's reported analgesic actions and could contribute to depression in reflex excitability of motoneurons.


ACTH4--10 antagonism of morphine-induced behavioral activation in the mouse. The 4--10 fragment of adrenocorticotrophic hormone (ACTH4--10; Met-Glu-His-Phe-Arg-Trp-Gly) was administered intracerebroventricularly to C57Bl mice with and without concurrent systemic injection of a standard dose of morphine sulfate. Morphine produced stereotyped behavioral activation (running, environmental oblivion, Straub tail) which was antagonized in a dose related fashion by the ACTH fragment. This extends the range of opiate--ACTH4--10 interactions to a novel species and behavioral model, and is consistent with previous findings.


Effect of L-glutamate and kainate on rat cerebellar cGMP levels in vivo. L-glutamate and kainate administered intracerebroventricularly (i.c.v.) both produced dose-dependent increases in rat cerebellar cGMP. The increased cGMP produced by L-glutamate, but not that produced by kainate, could be completely abolished by the glutamate antagonist glutamate diethylester which had no effect alone. The data suggest that L-glutamate and kainate may be acting upon different sub-populations of glutamate receptors, both of which may be involved in the regulation of cerebellar cGMP levels.


The effect of sleep upon the transmission of afferent activity in the somatic afferent system. Macaque monkeys were trained to fall asleep while sitting in a primate chair with their heads restrained. A gentle vibratory stimulus was delivered to the glabrous skin of the hand; it did not provoke awakening or change the sleep cycle of the macaque.  Postcentral neuronal response to the amplitude of a sine wave mechanical stimulus and neuronal spontaneous activity were observed repetitively during all the phases of normal night sleep cycles. One hundred six neurons which could be entrained by  a cutaneous mechanical stimulus were studied during both waking and sleep. At threshold, cyclic entrainment of the discharges of postcentral neurons decreased to 81 +/- 0.25% during light sleep (S1 + S2), to 64 +/- 0.26% during deep sleep (S3 + S4), and to 9 +/- 0.98% during desynchronized sleep with rapid eye movements (REMs).


Responses to stimulation of marginal fibers in the teleostean optic tectum. The marginal fibers (mf) constitute a major fiber component of the teleostean optic tectum, and this report deals with the physiological properties of these fibers and their postsynaptic elements. The mf are unmyelinated axons which originate at the torus longitudinalis and run lateralwards, parallel to one another, along the tectum's most superficial layer (stratum marginale). Here they synapse upon the dendritic arborizations of the pyramidal (p) neurons. These arborizations originate from a single apical dendritic shaft which, near the soma, receives retinofugal axon terminals. The p-neurons also have a basal dendritic shaft and a descending axon, both of which branch out horizontally at the stratum griseum centrale (SGC). The mf were stimulated through surface microelectrodes, and field potentials were recorded on-beam throughout the tectal thickness by means of micropipettes. The mf action potential (M-spike) may show two negative subpeaks which propagate at 0.20 and 0.16 m/s. Its refractory period is followed by a period of increased amplitude and decreased latency. The M-spike is followed by a series of slow waves, namely: (a) The S-wave, which probably represents the monosynaptic depolarization of the p-neuron's apical arbor; (b) The N-wave which possibly represents an active current sink at the point where these dendritic arborizations merge into the apical dendritic shaft; (c) The D-wave, recorded at the SGC, which possibly represents the activation of the p-neuron's axon and terminals (and perhpas also basal dendritic shaft and branches); and (d) the L-wave, which might represent a later depolarization of the p-neuron's apical arborizations. Morphologically and electrophysiologically, there are several similarities between the tectal mf/p-neuron system and the cerebellar parallel fiber/Purkinje cell system.


Glutamate and aspartate mimic the afferent transmitter in the cochlea. Glutamate (50 mM) and aspartate (50 mM) applied intracochlearly increase the spontaneous activity of ganglion cells of the auditory nerve. This activity may account for the decreased magnitude of the compound action potential observed with these substances in previous studies, and also may be additional evidence that these substances have a role in synaptic transmission between the hair cells and primary auditory afferent nerve cells.


Monocular deprivation and the signal transmission by X- and Y-neurons of the cat lateral geniculate nucleus. 1. In adult cats deprived monocularly from the second week of life, single neurons were recorded from the optic tract (OT), the lateral geniculate body (pars dorsalis, LGN) and the optic radiation (OR). The neurons were classified according to their visual response properties (Y/X) or their latencies to OT electrical stimulation (class I/II). 2. A close positive correlation (greater than 95%) was found between the visual classification and the latency classification (Y = I, X =  II). 3. The relative frequency of class I/Y-neurons was reduced in the group of pattern-deprived LGN neurons, but normal in the pattern-deprived OR neurons. 4. The ratio of the r1 and r2 wave amplitude of the OR-evoked potentials elicited by electrical stimulation of the normal or the deprived eye optic nerve was not affected by pattern deprivation. 5. The activity pattern of neurons recorded from the LGN or the OR did not differ in normal and pattern-deprived neurons belonging to the same class (on-center/off-center; Y/X). The same was true for the neuronal responses to electrical stimulation of the OT at different stimulus frequencies (1-200 stimuli/s). 6. The average maintained activity of pattern-deprived OT or OR neurons recorded while the eyelids of the deprived eye were still closed was either equal to or even somewhat higher than the average activity of the corresponding normal neurons. This was true when the patterned stimuli were presented stationary or were moved at random within the visual field simulating the effect of eye and head movements. The pattern deprivation effects found morphologically and physiologically in the visual cortex are, therefore, not induced by a diminished average activity of the input neurons from the deprived eye.


Locally evoked potentials in slices of rat neostriatum: a tool for the investigation of intrinsic excitatory processes. Field potentials, extracellular unitary discharges and intracellular potentials evoked by intrastriatal stimulation were recorded from slices (thickness 200-400 micron) of rat neostriatum maintained in an artificial medium. The field potentials consisted of two negative waves appearing at latencies of 0.5-1.5 ms (N-1) and 2-4 ms (N-2). Extracellular unitary records showed two typed of discharges, one with short but constant latencies at threshold level stimulation and the other with longer and variable latencies. In intracellular recordings the late discharge was seen to arise from EPSPs. Based on the intra- and extracellular unitary records, N-1 was identified as the population spike of antidromically or directly activated unitary discharges and N-2 as that of orthodromically activated discharges. This interpretation was substantiated by the fact that the N-2 potential was blocked in a perfusion medium containing a lower Ca++ or a higher Mg++ concentration than the standard solution. Neither interruption of ascending neostriatal inputs nor decortication 14 days prior to recording altered the configuration of the locally evoked potentials or the probability of synaptically driven discharge occurrence. Thus by intrastriatal stimulation, neostriatal neurons are activated antidromically or directly and/or orthodromically through intrinsic excitatroy synapses. Since the intracellular recordings showed that neostriatal neurons can be well preserved, this preparation can be regarded as a useful tool for electrophysiological and neuropharmacological investigations on intrinsic excitatory processes in the neostriatum.


Ultrastructural organization of thrombocytes in normal monkeys (Macaca arctoides) and in those with experimental viral malignant lymphoma. Data on platelet ultrastructure of 6 M. arctoides monkeys investigated before and during experimental viral malignant lymphoma are presented. It has been established that the inner structure of thrombocytes in healthy monkeys was almost similar to that of man. In experimental malignant lymphoma, irrespective of the clinical period of the disease, a decrease of alpha-, beta- and sigma-granules, a decrease of the functional activity of thrombocytes and an increase of delta-granules and glycogen granulation content were observed. The above mentioned disturbances can be a result of changes of megakaryocytes in malignant lymphoma. No viral particles in thrombocytes of neither healthy nor sick monkeys have been revealed.


Effect of various solubilizers on angiotensinII receptors in bovine adrenocortical plasma membranes. 3 nonionic detergents, Triton X-100, Lubrol WX and NP-40, inhibited binding of [3H]-ATII to bovine adrenocortical plasma membranes. This effect appeared to be direct and not due to solubilization of the ATII receptor by these agents. Sodium deoxycholate and the chaotropic ions, ClO4- and Br-, produced effects similar to the nonionic detergents.


Influence of alpha-MSH and ACTH on cortical bone remodelling in hypophysectomized rats. Hypophysectomy increases both periosteal resorption and endosteal apposition along the femur diaphysis in rat. Administration of alpha-MSH decreased the periosteal resorption but had no effect on the endosteal apposition. ACTH had only minor effects on the endosteum. Thus, alpha-MSH and ACTH, in the doses used, have different effects on cortical bone in rat. The effect of alpha-MSH on cortical bone could be an effect of the hormone alone or by its stimulation of other factors.


[Adenylate cyclase activation and its effects on intracellular cAMP in infected KB cells during trypsin-induced infectious Sendaï virus production (author's transl)]. KB cells infected by Sendaï virus can produce infectious virus if they are trypsinated twice over 24 h. Adenylate cyclase activity in infected KB cells is higher and more strongly activated by trypsin than that of control cells, but intracellular concentration of cAMP is the same, except during a short time after trypsinations, especially after the second trypsination which causes infectious virus production. During this short time, intracellular cAMP is slightly higher in infected cells. This miseffect of adenylate cyclase activation on intracellular cAMP concentrations might be related to an increased cell permeability caused by trypsin.


The effect of bisamidines of 2,6-diaminoanthraquinone on Entamoeba histolytica infections in rats and hamsters. Bisamidines of 2,6-diaminoanthraquinone have demonstrated potent activity against cecal and hepatic Entamoeba histolytica infections in rats and hamsters, respectively. A number of these compounds compared favorably, in overall drug efficacy, with metronidazole and other standard agents.


The anticonvulsant effects of propranolol and beta-adrenergic blockade. The anticonvulsant activity of racemic and (+)-propranolol was studied in rats. Neither drug changed the current to produce a minimal seizure in 50% of animals. Both drugs were effective in the maximal electroshock seizure test, the (+) isomer being more potent than the racemic form. Since the (+) isomer is practically devoid of beta-adrenergic blocking activity, the anticonvulsant effects of propranolol do not result from beta-adrenergic blockade.


Effects of [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) on cyclic 3',5'-nucleotide formation and phosphodiesterase activity. The orally-effective antiallergic compound [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) exhibited a potency equivalent to or 3 times less than theophylline in inhibiting guinea-pig lung and beef heart PDE, respectively, AY-25,674 did not affect the basal activity of guinea-pig lung adenyl cyclase. Although part of the antiallergic activity of AY-25,674 may be due to the ability to elevate cyclic AMP levels by PDE inhibition, other modes of action appear to be of greater relevance.


Effects of lithium chloride on peripheral acetylcholine release and brain acetylcholine levels in the guinea-pig. Lithium chloride administered acutely or chronically to guinea-pigs had no effect on brain level of acetylcholine or on peripheral release of acetylcholine from longitudinal muscle of the ileum. The results suggest differences between in vitro and in vivo action of lithium.


Ontogenetic changes in relative levels of cyclic AMP-dependent and cyclic GMP-dependent protein kinases in prostates, epididymides and testes from guinea-pigs. Changes in relative levels of cyclic AMP-dependent protein kinase (A-PK) and cyclic GMP-dependent protein kinase (G-PK) in prostates, epidymides and testes from guinea-pigs were examined at 3 different ages. During postnatal development, a decrease in the ratio of the 2 classes of protein kinases was seen in prostates, whereas increases of the ratios of the enzymes were found in epididymides and testes.


Prostaglandin-like substances in Propionibacterium acnes II. Stimulatory effect on ovarian cyclic AMP. The prostaglandin-like substances (PLS) from Propionibacterium acnes increased the ovarian tissue levels of cyclic AMP (cAMP) approximately 2-fold. The lipid material extracted from P. acnes thus behaved like PG's of the E-type, and since it is unlikely that other known stimulators of the ovarian cAMP system can be present in the bacterial lipid fraction, these experiments give further evidence in favour of the occurrence of PLS in P. acnes.


Decreased cyclic GMP levels in rat cecum mucosa during adaptive stimulation of Na-K-ATPase. In rat cecal mucosa, Na-K-ATPase specific activity and sodium and fluid absorption were increased by giving polyethylene glycol administration with the drinking water. Whereas cyclic AMP levels were unchanged, cyclic GMP was reduced by about 50%. This finding suggests a regulatory role of cyclic GMP in intestinal sodium and fluid absorption.


Zinc deficiency and lung converting enzyme activity in rats. Angiotensin converting enzyme activity was found to be significantly decreased in the isolated perfused lung from zinc-deficient rats when compared with that of controls. Addition of zinc ion to the superfusion medium did not cause a recovery in this decreased activity of the enzyme. It is postulated that zinc deficiency probably produces a structural change in the lung angiotensin converting enzyme.


Anticonvulsant activity of farnesylacetone epoxide--a novel marine natural product. A marine natural product, farnesylacetone epoxide, which is chemically related to juvenile hormone, has anticonvulsant properties at nonsedative doses in mice.


Reappearance of HCG-receptors in immature rat ovary after HCG-treatment is not due to receptor synthesis. In 24-day-old rats the reappearance of ovarian HCG-receptors after the injection of 200 IU HCG in independent of protein synthesis. The reutilization of occupied receptors in immature female rats is due to dissociation of the receptor-hormone complex.


[Substances having antiviral activity. IX. Synthesis and antiviral activity of 1-acyl-2-halo-3-formylindole thiosemicarbazones]. Some thiosemicarbazones of 1-acyl-2-chloro-3-formylindoles were synthesized and investigated for antiviral activity against vaccinia virus, HID stock and parainfluenza virus type 3 HA-I/CR-8 stock. Evidence of antiviral activity was found only against vaccinia virus, and was particular significant with the m-substituted 1-benzoyl-2-chloro-3-formylindoles. The first results of 2-substitution of chlorine by bromine in the indole skeleton, are reported.


[Comparative effect of psychotropic agents on orienting-motor responses and cerebral Na,K-ATPase activity in vivo]. Levomepromazine, chlorpromazine (10 and 30 mg/kg), etaperaxine, haloperidol (3 and 10 mg/kg) inhibited the exploratory-motor reactions of rats and the brain Na, K-ATPase activity an hour after their administration. The effects of tranquilizers as well as of antidepressants on the exploratory reactions and on the enzyme activity were not found to stand in a clearcut relation to each other. The stimulating effect of amphethamine (3 mg/kg) was accompanied by activation and suppressive action (10 mg/kg)--by inhibition of the enzyme. It is suggested that the inhibition of the brain Na, K-ATPase activity by psychotropic drugs may play a role in the mechanism of their sedative action.


A study of premarin intravenous and its influence on blood loss during transurethral prostatectomy. In a double-blind comparative study conjugated equine oestrogens (USP) (Premarin Intravenous) and saline were administered intravenously to patients undergoing transurethral prostatectomy. This paper proves that the placebo group (saline) displays a tendency, as indicated by two statistical tests, towards a significant increase in the red blood cells lost in the 24-hours after the operation. The Premarin Intravenous group showed no such tendency.


Autoantibodies to islet cells in diabetes mellitus. Numerous reports have confirmed the presence of islet cell antibodies in diabetic patients. These are found mostly in newly diagnosed insulin-dependent diabetic patients and in patients who have autoimmune polyendocrine disorders. Antibodies to beta cells, somatostatin, and glucagon-producing cells have been described as well. All these antibodies give strictly intracytoplasmic staining. It is therefore difficult to understand their role in the pathogenesis of pancreatic damage. The presence of another antibody is thus postulated.


Insulin secretion and cyclic adenosine 3', 5'-monophosphate levels in pancreatic islets of fed and fasted rats. Time course and dose kinetics during glucose stimulation. Incubation of pancreatic islets of fed rats at glucose 10 and 15 mM induced a rapid rise of the islet cyclic adenosine 3',5'-monophosphate (cAMP) content. Maximum levels were attained at 15 min and lasted until 30 min, after which cAMP declined again. Insulin secretion increased most rapidly from 15 min onward, i.e. after the rapid rise of cAMP. Islet cAMP at either 15 or 30 min showed its major concentration-dependent increase between glucose 7.5 and 10 mM. Glucose 15 mM did not further enhance the cAMP response, although this concentration increased insulin secretion more than two-fold over values observed at glucose 10 mM. Thus, the glucose dose-response relations for cAMP levels and insulin secretion appear to be different, indicating that factors other than cAMP alone determine the secretory response to glucose. Fasting for 24 and 72 h progressively inhibited glucose-induced insulin secretion. At glucose 15 mM the secretory inhibition disappeared after 30-45 min, but at glucose 10 mM it persisted for at least 90 min. Increasing periods of fasting also progressively delayed and inhibited the cAMP response to glucose, most strongly at glucose 10 mM. Fasting for 24 h shifted the glucose dose-response curves for cAMP and insulin secretion to the right, but the maximum responses at glucose 37.5 mM were not significantly inhibited. The secretory inhibition appeared to be linearly related with the cAMP content in two different ways: (a) At fixed concentrations of glucose, the increasing of the cAMP response (at 15 min) as induced by 24 and 72 h of fasting correlated with the secretory inhibition over the initial 30 min. (b) At one fixed period of fasting (24 h), the variable percent inhibition of the cAMP response to graded concentrations of glucose (5-37.5 mM) correlated with the percent secretory inhibition at these concentrations. These correlations were no longer apparent after 30 min of incubation. The results support the view that inhibition of the adenylate cyclase-cAMP system is a major determinant factor in fasting-induced impairment of insulin secretion during the initial 30 min of glucose stimulation.


Mononeuropathy in diabetes mellitus.

Mononeuropathy in diabetes mellitus. Fifty-one diabetic patients with mononeuropathies wereoneuropathy were studied to examine possible etiological factors, to determine the relationship with other diabetic complications, and to correlate with the presence and severity of background peripheral and autonomic neuropathy. The median, ulnar, and lateral popliteal nerves were most commonly affected and cranial neuropathy was relatively uncommon. When bilateral involvement of the same nerve was excluded, multiple mononeuropathies were found in only five patients. Median and ulnar mononeuropathy were gradual in onset and affected the dominant limb whereas other types of mononeuropathies were acute in onset with no predilection for either side. No consistent relationship was shown between the onset of mononeuropathy and age, sex, diabetic treatment, duration of diabetes, diabetic control, or other diabetic complications. In particular, there was no significant background peripheral and autonomic neuropathy, as assessed clinically and by objective tests, in almost one-half of the patients studied. It is concluded that diabetic mononeuropathy may occur independently of peripheral and autonomic neuropathy. It is possible, however, that a minimal degree of background damage, known to be present in all diabetic patients, may render them more susceptible than the general population to the various factors causing mononeuropathy.


An enzymecytochemical study on the adrenal of the freshwater teleost, Clarias batrachus (L.). The adrenal homologue of C. batrachus is distributed around the postcardinal vein in the pronephric head kidney. The cortical cells are round or oval in shape. They showed positive reaction for total lipid, glycogen and ascorbic acid. Their intense delta5-3beta HSDH activity indicates their capacity for steroid biosynthesis. In addition, the cortical cells of C. batrachus exhibited strong G-6-PD, NADPH diaphorase, NADH diaphorase, MAO and weak SDH and LDH activity. The presence of MAO suggests the aminergic control of the adrenal in this species and the silver positive fibres seen the cortical cells were hypertrophied, degranulated and the lipid content was also decreased. The chromaffin or medullary cells were distributed in groups among the cortical cells. They are largely oval or angular in shape. They react positively to ferric ferricyanide, chromaffin and argentaffin reactions and ascorbic acid test.


[Follow-up and management of patients with incomplete cone biopsies for dysplasias (author's transl)]. Report of 86 cone biopsies for dysplasias of the cervix. In 11 of these cases the cone biopsy did not show a clearly dysplasia free margin. These patients were followed from 1 to 6 years without further treatment and none showed a suspicious or positive pap smear. In young patients who desire further pregnancies it appears to be justified to follow without further treatment even after incomplete cone biopsies for dysplasia. Two patients with complete removal of the dysplasia and a normal margin in the cone biopsy later showed positive pap smears. The importance of further follow-up of the cases is stressed.


[Report on the clinical findings, the treatment, and the course of a case of hemangiofibroma of the adnexa (author's transl)]. The case of a hemangiofibroma of the right adnexa with locally infiltrative growth is described. The complete operative removal was not possible. A local recurrence was stopped from progressing by ligation of the most important arteries feeding the tumor. Progressive growth was stopped for two years and the patient wihch was an invalid became ambulatory following the operation.


[Modelling of a modification of chemical mutagenesis in human cells. II. The dependence of the effect on the time of culture treatment]. The influence of 2,3-aminopropylaminoethylthiophosphoric acid (2,3-APAETP) on the effect of the alkylating agent, thio TEPA, is investigated at different times of cultivation of human peripheral lymphocyte culture. The analysis of correlation equations shows that the results are described by the polynomes of 4-degree in variants treated with thioTEPA and 2,3-APAETP. The protector effect of 2,3-APAETP is determined not by the time from the culture stimulation but the time between the moment of culture treatment with 2,3-APAETP and thioTEPA and the moment of the fixation. It is shown that the points of maximal sensitivity of the cell cycle to thioTEPA and to the protective effect of 2,3-APAETP are similar.


Transformation of normal diploid cells by isolated metaphase chromosomes of virus-transformed or spontaneous tumor cells. Normal diploid human cells with a limited life-span in culture, as well as primary or secondary cell cultures of mouse or rat embryos, can be transformed in vitro (i.e. grow in soft-agar or low-serum medium) after a single exposure to metaphase chromosomes from SV40-transformed human or rat cells, Ad5-transformed human cells and several spontaneous human or mouse tumor cells. Chromosomes from normal diploid cells do not show any such transforming activity. As judged from the number of colonies formed in selective medium, the efficiency of transformation is, with some exceptions, of the order of 10(-5)--10(-6) and is generally higher for homologous than for heterologous transfers. A fraction of the colonies demonstrate abortive transformation. Nevertheless, using chromosomes from all but one donor cell population, at least one transferent cell line expressing a stable transformed phenotype has been established. Our results demonstrate that transformation of normal diploid cells by a presumptive chromosome-mediated gene transfer can be obtained with a variety of donor and recipient cells.


Hypercalcaemia, hyperphosphataemia and cytoplasmic inclusions in a patient with chronic lymphocytic leukaemia. The history of a patient with chronic lymphocytic leukaemia (CLL), known for ten years, is presented. During the terminal stage of the disease, she developed hypercalcaemia and hyperphosphataemia. The peripheral blood and lymph node lymphocytes contained crystalline inclusions in the dilated cisterns of the endoplasmic reticulum. The possible mechanism of hypercalcaemia and hyperphosphataemia and the significance of the crystalline inclusion are discussed.


Identification and characterization of melanin in tissues and body fluids. A new method for the unambiguous identification of melanin in biological materials has been developed. It may also be used to differentiate between melanins from various tissues and with various properties. The method is based on the detection and characterization of the free radicals in melanin by Electron Spin Resonance Spectroscopy. Applications of this approach include the identification of microscopically undetectable melanin in amelanotic melanomas and identification of the nature of the pigment in the Dubin-Johnson Syndrome.


The nucleoside phosphatase method as a marker for the recognition of the autochthonous plexuses in blood vessel walls and the possible connection of the latter with acupuncture mechanisms. The innervation of the blood vessels of the palatal mucosa of the frog is investigated histochemically. Besides vaso-motor nerves, fibers from the autochthonous plexuses also invest the vessels. Electron microscope studies of the latter show them to be non-nervous in structure contrary to previous belief. It is suggested they may influence vessels in a humoral manner and possibly play a part in acupuncture effector mechanisms. The electron micrographs are considered to reveal structure previously designated "terminal reticulum".


Contribution to the problem of the so-called nonpathogenic amoebae in the intestine of man. Among 10,418 patients of a Prague hospital, a plain infection with intestinal parasitic protozoans was identified in 1,319 persons (12.7%). Of these, 3.5% were  infested with Giardia intestinalis, 0.3% with Entamoeba histolytica forma minuta, 5.7% with Endolimax nana. We evaluated the frequency of findings of protozoans in various clinical diagnoses. A statistically significant increase in frequency was recorded for E. nana in diagnoses of eosinophilia, giardiasis, amoebiasis and helminthiasis. A slight increase above the average was found for Entamoeba coli in diagnoses of giardiasis and helminthiasis. Most cases of infection with Entamoeba histolytica were associated with a stay abroad. No increase in the frequency of these protozoans was recorded for patients with diarrhea. An analysis of the results indicated that a nonpathogenic amoeba might participate in the origin of intestinal disorders in man.


[Anti-D prophylaxis after abortions and interruptions]. In almost every pregnancy feto-maternal transfusions occur. From the 6th week of pregnancy, blood containing Rhesus antigens may be infused into the maternal circulation and cause sensitization. In case of miscarriage, abortion, ectopic pregnancy and cystic mole the chance of fetomaternal transfusion followed by sensitization of the mother is significantly increased. Therefore immunoprophylaxis with anti-D-immunoglobins should be performed in all these cases.


Antilipolytic nature of gut GLI, and mode of action of two highly potent intestinal lipolytic species in birds. Three major lipolytic factors, termed peaks 1, 2 and 3, according to their elution sequence from Biogel P6 columns, have been identified in duck intestinal extracts. The small molecular weight peaks 2 and 3, were even more lipolytically potent on chick adipocytes than pancreatic glucagon; peak 1, called gut GLI, because of its cross-reactivity in a radioimmunoassay for glucagon, modified the lipolytic activity of peak 2 and pancreatic glucagon. It did so by modifying their capacity to stimulate cyclic AMP production. Peaks 2 and 3 exert their lipolytic effects via different intermediary pathways: only peak 2 induced the formation of cyclic AMP. Insulin in birds is devoid of any antilipolytic activity; this fundamental role may be assumed by gut GLI.


Development of hyperlipidemia associated with increased lipolytic response of isolated adipose tissue cells following prolonged stimulation by an ectopic pituitary tumor. Implantation of MtT-F4 tumor, a mammotropic tumor that secretes large quantities of ACTH, GH and prolactin, into male Fisher rats induced the development of hyperlipidemia. Free fatty acid, triglyceride and cholesterol levels in the plasma were significantly increased at 31 days after tumor implantation. Blood glucose and glycerol levels remained normal, while uric acid concentration in the blood was significantly decreased. The lipolytic response of isolated adipose tissue cells to ACTH was significantly higher in cells derived from rats bearing an MtT-F4 tumor for 31 days than from their corresponding controls. However, the activity of adenylate cyclase in fat cells stimulated with ACTH was not significantly higher in cells derived from tumor bearing rats than in cells from control rats.


Isolation of the globular region of the subcomponent q of the C1 component of complement. Digestion after heat treatment of the subcomponent q of the C1 component of complement by collagenase leads to the isolation of the globular region of the protein. This product ('heads') is composed of three chains giving an overall molecular weight of about 57000. About half of the collagen-like region present in C1 q is lost after digestion. The 'heads' are shown to be soluble and hemolytically active products.


Covalent cross-linking of photosensitive phospholipids to human serum high density apolipoproteins (apoHDL). Human serum high density apolipoproteins were reassociated with three different lecithin species substituted with radioactively labelled photosensitive azido fatty acids, bis([3H]-16-azidopalmitoyl)-, bis([3H]12-azidooleoyl)- and bis([3H]18-azidolinoleoyl)glycerophosphocholine. The lipoprotein particles were reconstituted from a mixture of azido-labelled phosphatidylcholine and non-labelled dioleoylglycerophosphocholine (1:9). Excess lipid was separated from the homogeneous particles by Bio-Gel A-5m. The molecular weight, stoichiometry, fluorescence and circular dichroism of the reconstituted particles were determined before and after photoactivation with covalent cross-linking of the phospholipids with the apoproteins. The physical parameters of the reconstituted lipoproteins remained unperturbed by the cross-linking reaction between the generated nitrenes and apolipoprotein A-I and A-II. Thus the hydrophobic interactions of the phospholipid molecules with the apoproteins have been proved for the first time by a chemical method.


Guinea pig cell-mediated tumor immunity: the chromium release assay detects both cytolysis and serum blocking for syngeneic chemically-induced tumors. The chromium release test (CRT) was used to assess cell-mediated immunity to syngeneic, chemically-induced tumors in guinea pigs. The animal models were Sewall Wright strain 13 guinea pigs with 3-methyl-cholanthrene (MCA)-induced fibrosarcomas and strain 2 guinea pigs with diethylnitrosamine (DEN)-induced hepatocarcinomas. Regional lymph node cells were significantly cytolytic for the immunizing tumors, specifically so for three of the four tumors, and tumor-bearer sera could significantly block cytolysis. The two DEN-induced strain 2 hepatomas, line 1 and line 10, are antigenically distinct by the CRT but the two MCA-induced tumors have tumor specific antigens as well as a common, cross-reactive antigen.


Effect of chronic Trypanosoma brucei infection on the course of louping ill virus infection in mice. The course of louping-ill virus infection in mice chronically infected with Trypanosoma brucei was investigated. The mean survival time of mice infected with virus alone was 10.2 days, compared to 12.8 days in mice infected with T. brucei 14 days before virus challenge, and the cumulative mortality in the two groups was 62.5 and 90%, respectively. Virus was only inconsistently detected in the blood of mice given virus alone but was regularly present at high titers for up to 12 days in the blood of mice previously infected with T. brucei. Titers of virus in brains were also higher in dually infected mice, and encephalitis was of greater severity. These results indicate that the immunosuppressive effect of chronic trypanosomiasis may markedly increase susceptibility to acute virus infection. In addition, it is concluded that vector-vertebrate relationships of arthropod-transmitted viruses may be altered by concurrent trypanosome infection.


Lymphocyte proliferative response to viral antigen in pigs infected with transmissible gastroenteritis virus. Development and sequence of lymphocytes reactive to viral antigen in Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood of pigs orally inoculated with transmissible gastroenteritis virus were investigated by a lymphocyte proliferative assay. Lymphocytes reactive to the viral antigen were first detected in all the tissues of pigs tested on postinoculation day 7. Thereafter, they increased in proliferative reactivity and reached a maximal amount on postinoculation days 10 to 14. Antigen-reactive cells were persistently demonstrated in Peyer's patches and mesenteric lymph nodes for at least 110 days after inoculation, although lymphocytes decreased a little in reactivity to the viral antigen with the lapse of time. On the other hand, splenic and peripheral blood cells were found to have only transient proliferative reactivity. No antigen-reactive cells were detected in spleen or peripheral blood after postinoculation days 20 to 30. Lymphocytes decreased remarkably in reactivity to the viral antigen and phytohemagglutinin when treated with anti-porcine thymocyte serum and complement. Their reactivity to lipopolysaccharides was hardly affected by the treatment. Cells harvested on postinoculation days 45 to 60, however, responded a little to the viral antigen even after they were treated with anti-porcine thymocyte serum and complement. Lymphocytes reactive to the viral antigen and phytohemagglutinin belonged mainly to the erythrocyte rosette-forming cell fraction, whereas those reactive to lipopolysaccharides were mostly found in the rosette-nonforming cell fraction.


Persistence of avian oncoviruses in chicken macrophages. Inoculation of avian oncoviruses into 1- to 2-month old chickens led to a rapid production of antiviral humoral antibodies. Under these conditions it was found that avian leukosis viruses are sequestered in macrophages of peripheral blood, in which they can persist for a long period of time (up to about 3 years). In contrast, avian sarcoma viruses were never found in macrophages from chickens during the progression of sarcomas or after regression of the tumors.


Host defenses in herpes simplex infections of the nervous system: effect of antibody on disease and viral spread. BALB/c mice passively immunized with antibody to herpes simplex virus type 1 and challenged in the footpad with 10(5.7) plaque-forming units of herpes simplex virus type 1 were shown to be protected from neurological disease and death compared with control mice treated with normal serum or antibody to Sindbis virus. One hundred percent of untreated mice had virus recoverable from dorsal root ganglia by 48 h after infection. Whereas amputation of the infected limb at 48 h had no effect, antibody administration (resulting in titers of 1:8 and 1:16) was found to prevent acute neurological disease if administered no later than 48 h after infection. Antibody also restricted the extent of latent infection in the lumbosacral ganglia. The data provide strong evidence that antibody is effective in preventing spread of virus both in the peripheral nervous system and in central nervous system (spinal cord) tissue.


Susceptibility of lymphoblastoid cells to infection with human cytomegalovirus. Human lymphoblastoid cells of B- and T-cell origin were examined for their in vitro susceptibility to infection with human cytomegalovirus (CMV). Results of infectious-center assays, at virus to cell ratios of 10, indicated that in each of the lymphoblastoid cell lines tested less than 1% of the cells produced infectious virus. Under these conditions, CMV specific antigens were undetectable. Infection of lymphoblastoid cells with CMV resulted in atypical virus growth curves similar to those obtained with persistently infected human embryonic kidney cells. Although some variation existed in the relative sensitivity of lymphoblasts, cells of B (Raji, P3J-HR-1, RPMI 8226) and T (CCRF-CEM) origin were susceptible to infection with CMV. Variation in the sensitivity of lymphoblasts to CMV infection did not correlate with differences in virus adsorption or the presence of Epstein-Barr virus deoxyribonucleic acid. These studies suggest that human lymphoblastoid cells could serve as a model to examine persistent CMV infection in lymphoid cells of various origin.


Replication of herpes simplex virus type 1 in macrophages from resistant and susceptible mice. Studies were carried out to determine whether the in vitro capacity of adherent peritoneal cells to replicate herpes simplex virus type 1 (HSV-1) might correlate with the in vivo susceptibility of mice genetically resistant, moderately susceptible, or very susceptible to HSV-1 infection. Unstimulated and proteose peptone-stimulated monolayers restricted viral replication when infected immediately, but replicated HSV-1 when infected after 3 to 7 days of culture. Macrophages from resistant C57Bl/6 mice restricted HSV-1 replication significantly better than cells from susceptible mice. This function did not segregate with resistance, since macrophages from resistant F1 mice failed to restrict HSV-1 replication. Induction of peritoneal exudate cells with thioglycolate yielded cells capable of replicating HSV-1 when infected immediately after plating and after 4 days of culture.


Specificity of response to viral proteins in horses infected with equine infectious anemia virus. Three structural proteins of equine infectious anemia virus were purified, labeled with 125I, and utilized in radioimmunoassays with horse sera and antisera to heterologous retroviruses. Whereas radioimmunoassay titers for the major protein, p25, were 500- to 1,000-fold higher than titers in immunodiffusion, for clinical purposes these two procedures were equivalent. Antibodies to two low-molecular-weight proteins, p12 and p10, were also found in infected horses, but with a lower frequency and lower titers. As a rule, only sera positive for p25 also contained antibody to p12 and p10. Antisera to the major structural protein of other retroviruses did not precipitate equine infectious anemia virus p25. These sera include antibody to mammalian type C viruses, bovine leukemia virus, visna virus, mouse mammary tumor virus, squirrel monkey retrovirus, and Mason-Pfizer monkey virus.


Assessment of coxsackievirus B3 ts mutants for induction of myocarditis in a murine model. Ten temperature-sensitive (ts) mutants isolated from a myocarditis-inducing wild-type (WT) coxsackievirus B3 parent did not induce myocarditis in adolescent CD-1 mice. An avirulent prototype ts mutant from one of the three complementation groups adsorbed to murine cardiac tissue, as did WT virus. Heart tissues from mice inoculated with WT virus contained 100- to 1,000-fold more virus than heart tissues from mice inoculated with any of the three prototype ts mutants. WT virus exhibited a greater capsid stability and a higher efficiency of replication at 37 degrees C than any of the three prototype ts mutants. All three prototype ts mutants induced less interferon in vivo than WT virus. Cell-mediated immune responses, assessed by the cell migration inhibition assay, were different in mice inoculated with WT virus when compared to ts 5 mutant virus. Peritoneal exudate cells from mice inoculated with WT but not ts 5 virus reacted specifically against antigens in WT virus HeLa cell lysates and antigens extracted with KCl from cardiac tissues of mice inoculated with WT virus. Cardiac tissues of mice inoculated with WT but not ts 5 virus contained KCl-extractable antigens which were able to specifically inhibit the migration of peritoneal exudate cells taken from mice immunized with WT virus. Therefore, ts 5 neither elicited a measurable cell-mediated immune response nor induced antigens in cardiac tissues which were immunoreactive with sensitized-(WT virus)-peritoneal exudate cells. Of 9 revertant viruses isolated from the 10 ts mutants, 5 showed covariance in ability to replicate at 39.5 degrees C and capacity for induction of myocarditis. Some revertants exhibited a reduced capsid thermostability compared to WT virus but yet retained the capacity for induction of myocarditis. The data suggest that induction of myocarditis by coxsackievirus B3 variants depends on a combination of several variables, including capsid stability, capacity for replication at 37 degrees C, and expression of the three identified genes. All three prototype ts mutants served as vaccine viruses in preventing myocarditis in adolescent mice subsequently challenged with WT virus. However, all three prototype ts mutants and their revertant variants retained partial to complete lethality in CD-1 neonates.


Cellular interactions determine the rate and degree of interferon action. The rate and degree of interferon action on mouse embryo (ME), mouse L, and human amnion (WISH) cells were found to be dependent on the cell density. The most precipitous drop in interferon action occurred just below cell confluency. This effect was shown with both vesicular stomatitis virus and Sindbis virus and at both constant and variable input multiplicities of infection. At both "high" and "low" cell densities, cells attached to a surface develop viral resistance faster than suspended cells. These data indicate that either cell contact or close cell proximity is required for maximal interferon activity. These results are discussed in relation to interferon-induced transfer of viral resistance.


Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli. In the presence of Escherichia coli, myeloperoxidase-catalyzed oxidation of chloride ion resulted in formation of long-lived chloramine and/or chloramide derivatives of bacterial components. The same amount of these nitrogen-chlorine (N-Cl) derivatives was obtained with either hypochlorous acid (HOCl) or the myeloperoxidase system, indicating that myeloperoxidase catalyzed the oxidation of chloride to HOCl. Identical killing was obtained with HOCl or the myeloperoxidase system. About 30 to 50% of the oxidizing equivalents of HOCl were detected as N-Cl derivatives of peptides or peptide fragments that were released from the bacteria. The apparent molecular weight distribution of the peptides decreased with increasing amounts of HOCl, suggesting that peptides were fragmented by oxidative cleavage of chloramide derivatives of peptide bonds. The remaining 50 to 70% of the oxidizing equivalents of HOCl were rapidly consumed in peptide bond cleavage or the oxidation of other bacterial components. There was a close correspondence between the oxidation of bacterial sulfhydryls and bactericidal action. The N-Cl derivatives were lost and the oxidation of bacterial sulfhydryls increased over a period of several h at 37 degrees C. These changes were accompanied by increased killing. The increase in sulfhydryl oxidation and killing could be prevented by washing the bacteria to remove the N-Cl derivatives. Therefore, the N-Cl derivatives could oxidize bacterial components long after the myeloperoxidase-catalyzed oxidation of chloride was complete.


Murine neurotropic retrovirus spongiform polioencephalomyelopathy: acceleration of disease by virus inoculum concentration. A 10-fold reduction in the incubation period of murine neurotropic retrovirus spongiform polioencephalomyelopathy was effected by a 1,000-fold concentration of the cloned virus inoculum.


Mecillinam in urinary tract infections and in septicaemia. The clinical and bacteriological efficacy as well as the tolerance of mecillinam, a new beta-lactam antibiotic, administered parenterally in a dose of 40 mg/kg body weight, was investigated in 21 hospitalized patients with urinary tract infections or septicaemia. Success, defined as eradication of infecting organisms two to five days after treatment, was found in eight of 16 patients with urinary tract infections. Persistence of the original pathogen after treatment was seen in four patients, all with complicated urinary tract infection. Reinfection was seen in two patients, while the results were unevaluable in two cases. The five patients with septicaemia were all cured of their infection, this result being attributed solely to mecillinam in two cases, while a combined action of mecillinam and another antibiotic produced a cure in the other three cases. Escherichia coli resistant to mecillinam were isolated from two patients with persistence of bacteriuria following mecillinam treatment. Impairment of renal function may have been a contributory factor to the poor treatment response in patients with complicated urinary tract infection. Inasmuch as no important side-effects were recorded and mecillinam appears safe in patients with impaired renal and liver function, a higher dose may be indicated in these more complicated cases.


Rotavirus gastroenteritis in infants and young children. The electronmicroscopic examination of stool samples from 18 infants and young children with gastroenteritis, hospitalized at the Clinic of Infectious Diseases in Prague, was carried out. In ten children rotavirus was found in the faeces and the bacteriological findings were negative. Rotavirus particles were aggregated by convalescent child sera and by normal human gamma globulin. The clinical picture was characterized by the sudden onset of vomiting and fever, in one case with febrile convulsions. Diarrhoea was watery and yellow-green, and usually persisted for four to five days. The condition of the children improved rapidly after rehydration and a special diet. Older siblings, and in one family also adult members, were frequently affected.


Antigens in penicillin allergy. III. Antigen and antibody levels in mice treated with pure and contaminated penicillins. Using a radioimmunoassay, it was shown that commercially available ampicillin preparations often contain penicilloylated high molecular weight impurities. These possess immunological activities and stimulate penicilloyl-specific antibody formation in mice treated according to a therapeutic schedule. Using purified and experimentally contaminated preparations it was also found that exposure of the animals to Escherichia coli and Bordetella pertussis bacteria could increase the antibody formation to small amounts of impurities. In addition, penicilloylated antigen could be recorded in serum from treated animals. The antigen formed by penicilloylation in vivo, however, was very weak and did not induce much antibody formation when injected together with Freund's adjuvant in mice or rabbits.


In vivo suppression of allograft rejection by cyclic AMP increasing agents. This study was undertaken to elucidate the possibilities of modulating the survival of allogeneic tissue grafts by administration of cyclic AMP-increasing agents to the graft recipients. Heterotopic grafting of split mouse hearts across a strong H-2 barrier was used. It was found that Vibrio cholerae enterotoxin, which activates adenylate cyclase in mammalian cells, gave a marked prolongation of the graft survival time when given shortly before (but not after) transplantation. Also the administration of a combination of cyclic AMP and theophylline prior to or around transplantation increased graft survival time significantly. In contrast, no effect was obtained with choleragenoid, a membrane-binding cholera toxin analogue devoid of cyclic AMP-increasing activity.


Stimulatory and inhibitory effects of cyclic AMP on lymphocytes from atopic children. The effect of cholera toxin and dibutyryl cAMP on mitogen-activated lymphocytes from atopic and non-atopic individuals was studied. Cholera toxin enhanced stimulation by phytohemagglutinin of cells from small children but not from adults. Dibutyryl cAMP at low concentration (less than 10(-5) M) significantly enhanced the lymphocyte response to mitogens in some, but not all individuals. High concentrations, on the other hand, were consistently inhibitory. In atopic children, the lymphocyte response to T cell mitogen was significantly less stimulated by cholera toxin, and more inhibited by dibutyryl cAMP than the response of cells from non-atopic matched controls. Thus, T cells from atopic individuals appear to have an altered sensitivity to the action of cAMP, possibly resulting in an impaired balance between helper and suppressor T cells. The hypothesis is advanced, that such an altered balance is causally related to hyperproduction of IgE resulting in atopic disease.


Suppression of IgE antibody formation in mice: requirement for T-T lymphocyte interaction. Norman SWR mice injected with syngeneic spleen cells from ovalbumin (Ov)-primed mice were unable to make IgE anti-Ov antibodies when challenged with alum-pertussis-Ov. Immune T lymphocytes were shown to be responsible for the inhibitory effects of adoptively transferred spleen cells. Treatment of recipient mice with mild x-irradiation or with cyclophosphamide 2 or 3 days before cell transfer resulted in abrogation of the suppressor effect of immune cells. The injection of T lymphocytes into x-irradiated milce restored the suppressive effect of immune cells. It thus appears that immune T cells provide the stimulus for activation of suppressor T cells of the host. Although the generation of suppression is antigen-specific, the expression of suppression appears to be nonspecific.


Alkaline isomerization of horse and yeast cytochromes C. Spectrophotometric and circular dichroism studies. Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c. Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c. The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in amplitude of the negative circular dichroism band centered at 417 nm. This observation gives additional evidence that this negative band is closely related to the ligation of the heme iron by the sulfur atom of methionine 8u for each of the four haemoproteins examined.


Volume and polarity changes accompanied by amino acid substitutions in protein evolution. We evaluated the volume and polarity changes accompanied by amino acid substitutions along branches of the phylogenetic trees of cytochrome c, myoglobin and hemoglobin alpha and beta chains. In most cases the volume changes accompanied by the substitutions were found to be much larger than the volume of cavities existing in the interior of X-ray-analysed proteins. This implies that the interior of the proteins is very flexible and the necessary space for a larger amino acid residue substitution can be provided by adjusting nearby structures. Also, the volume and polarity changes are not particularly dependent on whether the substituted site is located in the exterior or interior of the proteins. This result supports the concept of the covarions by Fitch and Markowitz, when combined with the known fact that the exterior sites are more variable than the interior ones during protein evolution.


Isolation and primary structure of adrenocorticotropin from several species of whale. The isolation of adrenocorticotropin (ACTH) from fin (Balaenoptera physalus), sei (Blaenoptera borelis) and sperm (Physeter catodon) whale pituitaries is described. Their primary structures have been elucidated by the fluorescein isothiocyanate method and revealed to be identical to the structure of the human hormone. Fin whale exhibited an identical steroidogenic activity to that of the porcine hormone.


Purification and receptor binding properties of complexes between lutropin and monovalent antibodies against its alpha subunit. A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its alpha subunit, which is common to the glycoprotein hormones, has been purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex is heterogenous with respect to molecular size; 70--80% of the hormone is complexed with either two or three Fab fragments. The LH-Fab alpha complexes retain only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permits direct measurement of competition between receptor and the bovine complex because the alpha portion of the human hormone does not cross react significantly with antibodies directed against bovine alpha subunits.) Complex formation does not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-beta immunoassay which distinguishes free beta subunit from intact LH. Complexing of LH with Fab-alpha fragments also causes little or no change in the affinity of the hormone's beta subunit for anti-LH-beta antibodies indicating that significant changes in beta subunit conformation did not occur. The data show that at least two well-separated antigenic regions on the alpha subunit are exposed to the surface in the intact hormone. They are also in agreement with the proposal that the loss of binding activity to receptor is due to steric effects rather than to changes in conformation or dissociation, and that there may be sites on the alpha subunit which interact directly with the receptor.


[Generalized herpes simplex].

[Generalized herpes simplex]. This report is about a 58-year-old female patient with generalized herpes simplex virus exanthema. The virus was detected not only in the skin eruptions but also in rinsing liquid from the throat. Its typical feature revealed it to be herpes simplex virus Type I. On the basis of similar cases which have been described in literature, we point out that it is necessary to carry out virological and serological tests in cases of uncertain vesico-pustular or erythema exsudativum multiforme like exanthemas. These exanthemas must be separated from the postherpetic erythema exsudativum multiforme because of the identified virus.


[The effect of collagenase inhibition on bullous eruption and healing process in epidermolysis bullosa]. The collagenase inhibiting effect of erythromycin already observed in vitro was demonstrated also after oral administration of the drug in vivo in one child with epidermolysis bullosa letalis and one child with epidermolysis bullosa dystrophica. Despite inhibition of skin collagenase activity during administration of the drug the frequency of bullous eruptions and healing process of affected skin areas remained unchanged. This suggests no direct causal relation existing between skin collagenase activity and epidermolysis bullosa. Whether increased collagenase activity reflects a secondary reaction of the organism cannot be concluded from this study.