Toxicity and residue studies in dairy animals with firemaster FF-1 (polybrominated biphenyls). Two studies (one in Holstein calves and one in Holstein cows) were conducted to determine potential toxicity and residue levels following oral ingestion of polybrominated biphenyls (PBB). The material was FireMaster FF-1. Administration was by gelatin capsules. Doses in calves were 0.1, 1.0, 10, or 100 mg/kg body weight, while doses in cows were equivalent to 0.01, 0.1, 1.0, or 10 ppm in the diet. The calves were sacrificed after 2, 4, 6, or 12 weeks. The cows were fed 158 or 228 days, and were then in a recovery period for 182 or 112 days. In the calf study, signs of toxicity were observed only in animals fed 100 mg/kg-day. Administration of 10 mg/kg-day or less for up to 12 weeks caused no overt signs of toxicity. Histologic studies were conducted upon selected organs and tissues taken at time of sacrifice. The only treatment-induced lesions among animals fed 0.1 mg/kg-day were minimal lesions in the kidney and skin in the one calf fed at this level for 12 weeks. Treatment-induced lesions were present in the kidneys, skin, and/or liver from some animals fed levels of 1.0 mg/kg-day and above. The relative severity of these lesions was related to the level and length of exposure. Treatment-associated changes were observed in the testes of all males in this study. The hypospermatogenesis observed was consistent with the age of the animals due to prepuberal development of the testes. No clinical signs of toxicity or histologic changes attributed to PBB were observed in the cows. Two cows were pregnant at the initiation of the study and give birth to normal, healthy calves during the study. These calves grew normally and appeared healthy when sacrificed at about 6 months of age. Residue levels were quantitated as the hexabromobiphenyl isomer (BP-6) which is the major isomer present in the PBB mixture. Tissue residue levels in calves increased with dose and duration of administration of PBB with highest levels being found in the fat. At 100 mg/kg the levels in fat were about 6000 to 6300 ppm after 6 to 12 weeks. Residue levels of BP-6 in milk and fat of the cows also increased with dose and duration of administration. Maximum levels in milk were about 1/3 the levels in the diet. In general, the levels plateaued after about 4 to 6 weeks and did not go appreciably higher even though administration of the FireMaster FF-1 was continued. Maximum levels in fat were on the order of approximately 4.5 times the levels in the diet, except at the lowest dose level. Residues decreased in milk after administration of PBB was discontinued, but detectable levels were still present 6 months later. Residues were found in the calves born of treated cows indicating passage of the PBBs through the placental barrier and/or ingestion through the milk.


Effects of polybrominated biphenyl on milk production, reproduction, and health problems in Holstein cows. PBB found in relatively low levels among animals present on a cross-section of Michigan farms during the time PBB was inadvertantly added to dairy feeds had no effect upon these animals' milk production, body weight, weight gain, breeding and reproduction performance, incidence of commonly experienced health problems, calving rate, and the health of their calves. No significant differences in these vital areas could be seen between Michigan animals exposed to PBB and equivalent Wisconsin animals which had not been exposed to PBB when both groups were subjected to equivalent management practices. No pattern of gross of histopathological lesions was seen upon necropsy between test animals and control animals.


Limited tryptic digestion of human serum low-density lipoprotein: isolation and characterisation of the protein-deficient particle and of its apoprotein. Limited tryptic digestion of human serum low-density (LD) lipoprotein (rho 1.024-1.045 g/ml) under defined conditions permitted isolation by gel filtration chromatography of a stable, protein-deficient lipoprotein; the liberated protein was separated as a mixture of peptides of low molecular weight (less than 5000). Comparison of the chemical, physical and immunological characteristics of the trypsin-treated LD-lipoprotein with those of the native preparation revealed several differences, including (a) a diminished protein content (loss of some 20-25% of the total protein of LD-lipoprotein) and increased proportions of the various lipid components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD-lipoprotein (d) an increased net negative charge; and (e) a partial immunological identity between LD-lipoprotein and the corresponding trypsin-treated fraction. While the amino acid compositions of the protein moieties of LD-lipoprotein and of trypsin-treated LD-lipoprotein were essentially identical, trypsin-treated apo-LD-lipoprotein was distinct in its complete solubility in urea-containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents. Comparison of the apoproteins of the native and trypsin-treated LD-lipoproteins by electrophoretic techniques based on molecular weight revealed a transformation of the high-molecular weight material (greater than 250 000) characteristic of apo-LD lipoprotein into several polypeptide species (10 major forms) ranging in size from 161 500 to about 10 000. The largest of these (band b1: 161 500) could be completely dissociated into smaller components (b2: 93 500 and b3: 77 000) upon extensive heat treatment at 90 degrees C. Electrophoresis of the soluble fraction of apo-LD-lipoprotein and of that from its trypsin-treated counterpart in polyacrylamide gels containing urea at basic pH showed the disappearance of the small amounts (less than 5%) of C apoproteins of apo-LD-lipoprotein upon tryptic treatment. These results, which were highly reproducible in LD-lipoprotein preparations from different individuals, suggest that trypsin-treated LD-lipoprotein may provide a model for investigation of the organisation and structural role of the prinicipal apoprotein (apolipoprotein-B) in the LD-lipoprotein molecule.


Structural studies of iron and cobalt tetrasulfonated phthalocyanine-globin complexes. The structure of the complexes of iron and cobalt tetrasulfonated phthalocyanines with globin has been investigated by circular dichroism (CD), electron paramagnetic resonance (EPR) and polyacrylamide gel electrophoresis. Electrophoretic investigations and the molecular weight estimation indicates that the model complexes in the solutions are dimers. It is evident from the results of CD measurements that the incorporation of the iron or cobalt tetrasulfonated phthalocyanine into apohemoglobin significantly increases the helical structure of the protein and causes an appearance of the induced Soret and visible Cotton effects. Unlike methemoglobin, several discrete transition energies in the CD Soret band of Fe(III)L-globin are observed which suggest an inequivalence of the subunits within this complex. This suggestion is supported by EPR studies, which show that the iron atoms in Fe(III)L-globin are in two low electronic states. Electronic structures of the cobalt ions in Co(II)L-globin and oxyCo(II)L-globin are similar to those of coboglobin and oxycoboglobin, respectively, as is proved by EPR results. On this basis we conclude that the oxygen adduct of Co(II)L-globin can be described as a superoxide ion corrdinated to a formally cobaltic phthalocyanine compound.


Refolding and reactivation of liver alcohol dehydrogenase after dissociation and denaturation in 6M guanidine hydrochloride. Horse-liver alcohol dehydrogenase has been dissociated and denatured by 6 M guanidinium hydrochloride. Removal of the denaturant under optimum conditions of the solvent leads to partial reactivation. The concentrations of the enzyme, as well as the coenzyme (NAD+), and Zn2+, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products. Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation-association provide maximum yields (approximately 70%). The sigmoidal kinetic traces suggest a superposition of first-order transconformation and second-order association reactions; the latter are corroborated by the concentration dependence of the reactivation reaction. The coenzyme, NAD+, has no influence on the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of reactivation. The process of renaturation, as reflected by the regain of native fluorescence shows complex kinetics: rapid relaxations are followed by slower first-order and second-order processes which parallel reactivation.


Occurrence of secondary cystathioninuria in children with inherited metabolic disorders, liver diseases, neoplasms, cystic fibrosis and celiac disease. Secondary cystathioninuria was found in two of 46 children suffering from tumors, leukemia, liver disease, inherited metabolic disorders, cystic fibrosis and celiac disease. Of these two patients, one had congenital biliary atresia and the other cytomegalovirus infection. Seven further children had only moderately elevated excretion of cystathionine. It is suggested that secondary cystathioninuria is uncommon in the disease investigated.


Superior orbital fissure syndrome. Review of 130 cases. Superior orbital fissure syndrome is a symptomatologic complex, consisting of retroorbital pain, paralysis of extraocular muscles, impairment of first trigeminal branches and frequent involvement of the optic nerve. From a review of 130 published cases including two personal observations, it appears that the clinical subdivisions and the several eponymic differentiations of this syndrome are unjustified on the basis of etiologic, therapeutic and prognostic elements. Only the presence--or absence--of optic signs may allow to hold the clinically useful distinction between the often 'benign' superior orbital fissure syndrome without optic nerve involvement, and the orbital apex syndrome where orbital exploration may be recommended. This review concludes that repeated neurosurgical and neuroradiologic investigations--with the exceptions of carotid angiography and orbital phlebography--can be avoided in such cases.


Nerve conduction velocity of small components in human sensory nerves. Studies in normal and diseased nerves. Slow conducted components of sensory nerve action potentials were investigated in median and in sural nerves of controls and in patients with peripheral nerve diseases. In the normal group the slow components showed no relation to age which is in contrast to the maximum velocity. In both the median nerve and sural nerve of about 20% of the patients with neuropathy exclusively a decrease of the conduction velocity of slow components was found, the other investigated parameters were normal. Possible causes are an alternation of the myelin sheath or changes in the properties of the nodal gap membrane.


Homovanillic acid and 5-hydroxyindoleacetic acid in lumbar cerebrospinal fluid after total and REM sleep deprivation in humans. Lumbar CSF HVA and 5-HIAA levels were assayed in 3 groups each of 10 subjects, which were respectively deprived of sleep for 30 h, deprived of REM sleep and disturbed with several awakenings during SW sleep for two consecutive nights. HVA levels after total sleep (39 +/- 20 ng/ml) or REM (35 +/- 11 ng/ml) deprivation as well as after SW sleep awakenings (32 +/- 26 ng/ml) were not different from controls (42 +/- 14 ng/ml). 5-HIAA levels after REM deprivation (32 +/- 15 ng/ml) appeared increased when compared with controls (21 +/- 7 ng/ml), total sleep-deprived subjects (21 +/- 10 ng/ml) or subjects with SW sleep awakenings (27 +/- 13 ng/ml). Possible increase in 5-HT turnover after REM deprivation and possible 5-HT role in REM sleep regulation in humans are discussed.


Prazosin and presynaptic alpha-receptors in the cardioaccelerator nerve of the dog. In vagotomized, spinal-sectioned dogs, prazosin and phentolamine enhanced positive chronotropic responses to cardiac accelerator nerve stimulation. In additional dogs the inhibition by clonidine of heart rate response to continuous accelerator nerve stimulation (presynaptic effect), and the vasopressor effect of clonidine (post-synaptic effect), were antagonized by prazosin, phentolamine and yohimbine; cumulative doses for 50% antagonism of the cardiac chronotropic effect were 103, 50 and 13 microgram/kg i.v., respectively, and those for 50% antagonism of the vasopressor effect were 39, 38 and 3 microgram/kg i.v., respectively. On isolated rabbit pulmonary artery, prazosin antagonized electrically evoked contractions but had no effect on 3H output, whereas yohimbine enhanced both. The results indicate that prazosin, like phentolamine and yohimbine, blocks presynaptic alpha-adrenergic receptors on the cardiac accelerator nerve of the dog but, unlike yohimbine, prazosin does not block these receptors on noradrenergic nerves of rabbit pulmonary artery. It is concluded that the relative activity of a compound at pre- and postsynaptic alpha-receptors is not the same for all organs. The results are discussed relative to the fact that prazosin causes hypotension without significant reflex tachycardia in dog and man.


Increased noradrenaline release in rat portal vein during sympathetic nerve stimulation due to activation of presynaptic beta-adrenoceptors by noradrenaline and adrenaline. With the aim of investigating whether exogenous noradrenaline (NA) and adrenaline (A) can modulate transmitter release via the stimulation of presynaptic beta-adrenoceptors, 3H-release from isolated portal veins was studied after pretreatment with 3H-1-NA, phenoxybenzamine, desipramine and normetanephrine. NA (10 muM) and A (0.05 muM) increased the fractional 3H-release elicited by sympathetic nerve stimulation by 30%. This effect could be blocked by d, 1-propranolol which per se reduced the release by 10%. It is concluded that NA can facilitate its own release via a presynaptic beta-adrenoceptor-mediated positive feed-back mechanism and that adrenaline can stimulate this beta-adrenoceptor-mediated mechanism.


Specific antagonism of excitant amino acids in the isolated spinal cord of the neonatal rat. The specificity of the neurodepressant actions of D-alpha-aminoadipate, alpha,epsilon-diaminopimelic acid, HA-966 (HAP) and Mg2+ has been investigated. On the isolated spinal cord of the neonatal rat, ventral root depolarizations produced by kainate, substance P, carbachol and noradrenaline were relatively unaffected by the same concentrations (0.25--1 mM) of the agents as those which reduced synaptic activity and ventral root depolarizations produced by N-methyl-D-aspartate (especially), L-aspartate and L-glutamate. The same or higher concentrations of the agents did not affect excitatory transmission in the isolated rat superior cervical ganglion. It is proposed that the agents specifically block synaptic transmission mediated by an excitatory amino acid.


Convergence of cerebral inputs onto dentate neurons in monkey. The patterns of convergence of inputs from different areas of the cerebral cortex and the peripheral nerves onto single dentate neurons was studied in cebus monkeys. Dentate neurons receive their strongest and most numerous inputs from the premotor and supplementary motor regions of area 6. The sensorimotor and frontal cortices have weaker projections to the dentate nucleus, while peripheral nerves and many other association cortical areas were found to be effective in influencing cells of the lateral cerebellum. Dentate cells that respond to stimulation of hindlimb regions of the sensorimotor cortex tend to receive their principal input from the supplementary motor area and medial premotor regions, while neurons responding to forelimb sensorimotor cortex tend to receive lateral premotor inputs. In addition there is a topographical organization within the ventral pole of dentate with the hindlimb represented in the anterior regions and the forelimb in the posterior regions. These results are compared with those of similar studies of interpositus and dentate neurons in cat and monkey. The differences between the afferent inputs to dentate and interpositus are consistent with the suggestion that the lateral cerebellum is involved in programming movement parameters before movement initiation while the intermediate zone is involved in up-dating the evolving movement.


Properties of a new vestibulospinal projection, the caudal vestibulospinal tract. Neurons in the caudal portions of the medial and descending vestibular nuclei and in vestibular cell group f that project to the cervical or lumbar spinal cord were located by antidromic spinal stimulation. These caudal vestibulospinal tract (CVST) neurons have a median conduction velocity of 12 m/sec, which is well below the conduction velocities of typical lateral or medial vestibulospinal tract (LVST, MVST) axons. The descending fiber trajectories of CVST neurons, determined by comparing thresholds for activation of each neuron from six points in the spinal white matter, were remarkably diverse. Unlike LVST and MVST axons, which are located in the ipsilateral ventral funiculi, CVST axons can be found in both the ventral and dorsolateral funiculi on both sides of the spinal cord. The CVST system is thus both anatomically and physiologically different from the LVST and MVST.


Functional organization of the corticofugal system from visual cortex to lateral geniculate nucleus in the cat (with an appendix on geniculo-cortical mono-synaptic connections). 1. In the cat visual cortex (VC), electrophoretic glutamate application at a depth corresponding to layer VI may have excitatory or inhibitory effects on relay cells of the lateral geniculate nucleus (LGN). Corticofugal excitation was seen, if the receptive field centers (RFCs) of the VC neurons recorded at the application site were within 2.3 degrees of the RFCs of the LGN neurons under test. Inhibitory effects were seen if the RFCs of both cells were further apart up to 3.1 degrees. Glutamate application at more superficial cortical sites had no effect on LGN-neuron activity. 2. Cross-correlation analysis between spontaneous activities of simultaneously recorded VC and LGN neurons revealed excitatory cortico-geniculate connections in 18 pairs with RFCs separated by less than 1.7 degrees. In 15 pairs the peak latency of the excitation was 2--5 msec (3.4 msec in the average), 3 pairs showed long cortico-geniculate latencies (13--18 msec). The existence of a fast and slow cortico-geniculate system is suggested. 3. Inhibitory cortico-geniculate interaction was demonstrated with cross-correlation analysis in 8 pairs of which 4 had RFCs separated by more than 1.7 degrees. The onset latency of the inhibition was 2--7 msec except for 2 pairs with about 20 msec latency. 4. Most of the LGN neurons which were affected by cortical glutamate application or which showed an excitatory or inhibitory connection with a VC neurons were sustained cells, while the majority of VC neurons which were recorded in the effective glutamate application sites or which showed a significant interaction with LGN neurons in the cross-correlogram were binocularly driven and complex, with mostly large RFCs (mean diameter 3.5 degrees). They responded briskly to moving small spots as well as to moving slits. 5. It is concluded that the corticofugal excitatory effect is transmitted through monosynaptic links from VC neurons located in layer VI (complex cell) to LGN relay neurons (mostly sustained-cell) and this system is organized in a precise topographical manner. 6. In an Appendix neuron pairs which showed a positive correlation in the geniculo-cortical direction were described. The findings may support the view that complex as well as simple cells are drive monosynaptically from geniculo-cortical afferents of the sustained or transient type.


Vestibulo-ocular reflex from the posterior canal nerve to extraocular motoneurons in the cat. In the anesthetized cat, the posterior canal nerve (PCN) was stimulated by electric pulses and synaptic responses were recorded intracellularly in the three antagonistic pairs of extraocular motoneurons. Pure reciprocal effects were obtained in the motoneurons innervating the antagonistic pair of ipsilateral oblique muscles and the antagonistic pair of contralateral vertical rectus muscles. These responses consisted of low threshold disynaptic excitatory postsynaptic potentials (EPSPs) in either the contralateral superior oblique (c--SO) (trochlear) or contralateral inferior rectus (c--IR) motoneurons and of disynaptic inhibitory postsynaptic potentials (IPSPs) in either the ipsilateral inferior oblique (i--IO) or ipsilateral superior rectus (i--SR) motoneurons. In addition, disynaptic IPSPs were also found in (i--SO) motoneurons. Mixtures of low threshold (di or trisynaptic) EPSPs and IPSPs were found in all other extraocular motoneurons except for the contralateral lateral rectus (c--LR) motoneurons. These results may afford a basis for the characteristic eye movements induced by vertical canal nerve stimulation.


Murine cytomegalovirus and fertility: potential sexual transmission and the effect of this virus on fertilization in vitro. Male mice were inoculated with murine cytomegalovirus (MCMV) to produce an acute generalized infection. Infections virus was recovered from both epididymal sperm and seminal vesicles as well as from uterine sperm collected from mated females, suggesting that MCMV might be transmitted sexually. Because the presence of virus in the ejaculate might affect the fertilization process, the effect of MCMV on the fertilization of mouse gametes and on subsequent embryonic development was studied in vitro. Although the fertilization rate was reduced when sperm were preincubated with infectious virus, this was also the case when heat-inactivated virus was used, leading to the conclusion that this effect was not due to a direct infectious interaction between virus and gametes. Subsequent embryonic development was normal, and there was no evidence of productive infection of the preimplantation embryo.


[Participation of thyrocalcitonin in the development of stress]. The immobilization stress in rats entailed raise of calcium content in the plasma followed by a decrease to the hypocalcaemic level. The thyrocalcitonin activity of plasma was undetectable in intact rats, but increased to 5 mU/ml after 3 hrs of stress. Administration of bovine thyrocalcitonin (10 U/100 g) before the immobilization inhibited the lowering of CRF activity of hypothalamic extracts, ACTH content of hypophysis, and ascorbic acid concentration in adrenal glands of the rats under stress, i.e. inhibited the development of stress reaction.


[Reactions of the microcirculatory bed of cat mesentery during exposure to heat]. The TV-microscopy in cats revealed that, by the 30th minute of 2-hr overheating, the arterioles are dilated and venules constricted. In the course of raise of body temperature from 36 degrees to 40 degrees C, the number of dilatory and constrictory responses of venules remains the same while the number of arteriole dilatory responses diminishes and of constrictory responses increases. At 40 degrees C of body temperature, the number of dilatory responses of the mesenteric arterioles and venules makes about 20--30% while the number of constrictory responses--70--80%. The extent of dilation of arterioles and venules increases with the heating while the extent of constriction, having achieved a certain level, starts to diminish. The greatest responses occurred in the microvessels with initial diameter 60 and more microns. The responses of microvessels with a lesser diameter could be different. The data obtained using hexonium, dihydroergotoxin, inderal, atropin, dezeril, methyamide, and tavegil, suggest participation of adrenergic and histamingergic substances in vasomotor responses of the microvessels to the hyperthermia.


Cyclic AMP levels in isolated frog skin epithelium: effects of phosphodiesterase inhibitors, oxytocin and catecholamines. Direct measurements of cyclic AMP were performed in the isolated epithelium of frog skin. Phosphodiesterase inhibitors (methylxanthines, papaverine) and activators of adenylyl cyclase (oxytocin, catecholamines) significantly increased the cyclic AMP content. Propranolol completely blocked the generation of cAMP induced by beta-adrenergic agonists but had little or no effect on that induced by oxytocin. Phentolamine enhanced the cAMP production by adrenalin and noradrenalin. At supramaximal concentrations, oxytocin and isoproterenol produced similar increments in cAMP, while exposure to both agents roughly doubled the increase in cAMP. The results suggest the presence of independent receptors for oxytocin and catecholamines in frog skin, with additive effects on cAMP generation.


Nuclear triiodothyronine receptors in the developing rat brain. This study examines whether the high sensitivity of the developing brain to thyroid hormones and the purported decline in sensitivity in adulthood, are correlated with changes in the density and affinity characteristics of specific nuclear T3 receptors. The authors have found that the nuclei of cerebral hemispheres have a high density of T3 receptors at birth (212 +/-28 X 10(-17) mol/microgram DNA) which declines to adult levels by the end of the second postnatal week (115 +/- 7 X 10(-17) mol/microgram DNA), remaining at this level until 6 months of age. Even though no significant changes were detected in the equilibrium dissociation constant (Kd) during the early period of development, comparison neonatal with the adult brain reveals a decrease in Kd (neonatal, 3.9 X 10(-10) M; adult, 2.3 X 10(-10) M). In the developing animal, neonatal thyroidectomy increased the number of binding sites in the nucleus by 36--44%. It is concluded that the high number of nuclear T3 receptors in the first week of postnatal life is correlated with the high dependence of brain tissue on thyroid hormones and that the decline in brain sensitivity may be associated with the decline in nuclear T3 receptors. The high affinity and density of nuclear receptors in adult brain tissue relative to the developing brain and liver, respectively, point to a continued regulatory role of thyroid hormones in brain.


HCG-induced changes in the number of rat testis LH/HCG receptors. Adult male rats were injected subcutaneously with a mixture of hCG and [125I]hCG (3, 5, 10, 100 or 1000 IU), and killed 3, 6, 16, 24 or 48 h after injection. Specific uptake of [125I]hCG by the testes as well as the remaining in vitro binding capacity were measured. Cycloheximide (1.5 mg/kg) was given together with hCG to another group of animals. The effect of hCG on testicular LH/hCG receptors was found to consist of two phases: first, 6--16 h after injection, a significant increase in the binding capacity occurred, which was found with the lower 3--10 IU doses of hCG. Thereafter there was a pronounced decrease in receptor concentration, which was directly correlated to the dose of hCG. Experiments with cycloheximide suggest that the decrease in binding, but not the increase, is at least partly dependent on protein synthesis.


Comparison of alpha-ketoisocaproic acid and glucose in rats: effects on insulin and somatostatin release and on islet cAMP content. The insulinotropic effects of alpha-ketoisocaproic acid and glucose reveal many common characteristics in vivo and in vitro. They qualify as initiators of insulin release, their action is amplified by potentiators of insulin release, and they have a similar potency at equimolar concentrations. The dynamics of insulin release evoked by alpha-ketoisocaproic acid and glucose are similar. Epinephrine completely inhibits the insulinotropic effect of glucose and alpha-ketoisocaproic acid. Mannoheptulose exhibits a complete, immediate and reversible blockade of glucose-induced insulin release. In contrast, inhibition of alpha-ketoisocaproic acid-induced insulin release occurs after a lag period and is not reversed by removal of the inhibitor. alpha-ketoisocaproic acid, at equimolar concentrations, is several-fold more effective than glucose in elevating cAMP content in islet. alpha ketoisocaproic acid and glucose are about equally effective in stimulating somatostatin release from isolated rat pancreatic islets. This stimulation is inhibited by epinephrine. Mannoheptulose inhibits only somatostatin release induced by glucose but not by alpha-ketoisocaproic acid. It suggested that the insulinotropic characteristics of glucose and alpha-ketoisocaproic acid reveal many common features, while their mode of action appears to be different.


LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation. Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.


Receptor regulation and target cell responses: studies in the ovarian luteal cell. Studies of hormone-induced 'desensitization' in the luteinized rat ovary show that changes in receptor number and adenylate cyclase at the cell surface result in an altered biological effect of luteinizing hormone on progesterone production. The sensitivity of these effects to changes in gonadotropin levels suggests that receptor turnover or processing is involved in the normal mechanism of hormone action.


Topographical studies on histamine- and adrenaline-sensitive adenylate cyclases in gastric and duodenal mucosa of human beings. The distribution of histamine- and catecholamine-sensitive adenylate cyclases in human gastric and duodenal mucosa was studied. Basal enzyme activities averaged 155 pmol cAMP/mg prot./15 min in fundic gastric mucosa, 305 pmol cAMP/mg prot./15 min in the antral and 344 pmol cAMP/mg prot./15 min duodenal mucosa. 1 mM histamine induced a more than 2-fold increase of enzyme activity in fundic homogenates, whereas this secretagogue was nearly ineffective in similar preparations from the antral region (1.2-fold increase of enzyme activity). The response towards adrenaline was virtually identical in fundic and antral mucosa preparations. The duodenal enzyme was insensitive toward this catecholamine as well as to histamine. The data are suggestive for a messenger function of cAMP in histamine-stimulated gastric acid secretion.


Effect of the association time of in vivo bound prolactin on the [125I]prolactin receptor assays of female rat livers. Significantly (P less than 0.01) reduced 125I-labeled ovine prolactin binding (mean, --22%), as a result of an ether anesthesia-reduced rise in serum prolactin, was observed in plasma membrane preparations of liver samples of female rats taken after 5 min of etherization when compared to samples taken from the same animals during the first minute of etherization. This reduction in assayable receptors occurred after 1 h but not 2 h of assay incubation time. Significantly (P less than 0.05) reduced 125I-labeled ovine prolactin binding (mean, --16%) was also observed in liver samples exposed to a 30-min ether-induced rise in serum prolactin when compared to liver samples taken during the first minute of etherization. In contrast, this reduction was apparent at assay incubation times of 1, 2 and 4 h but not at 10 h. These results suggest that serum prolactin can bind to prolactin receptors in vivo and partially block subsequent 125I-labeled ovine prolactin receptor assay. In addition, these data provide evidence that a complex time-dependent binding of prolactin may occur in the plasma of the female rat liver.


Evidence of agar gel electrophoresis changes of lipoprotein-X after phospholipase A and deoxycholic acid. The effect of phospholipase A from snake venom and deoxycholic acid on lipoprotein-X (LP-X) recovered from the cathode side of a previous agar gel electrophoresis is described. Adding phospholipase A and deoxycholic acid to the removed cathodal fraction is followed by a marked migration to the anode side on a second electrophoresis procedure. This seems to confirm that phospholipase A and bile salts on LP-X particles modifying their agar gel electrophoretic migration characteristics.


Rat testis LH/hCG recptors and testosterone production after treatment with GnRH. Adults male rats were implanted subcutaneously for 1, 2 or 4 days with Alzet osmotic minipumps, infusing synthetic gonadotropin-releasing hormone (GnRH) at a rate of 0.1 microgram/h. Plasma LH increased significantly, reaching a maximum after 2 days treatment. Plasma and testicular testosterone levels increased to a maximum in 1 day, and decreased gradually thereafter, reaching the control level by day 4. Testicular specific [125I]hCG binding decreased gradually during the treatment, being 57% of the controls by day 4. It is concluded that changes of physiologic magnitude in circulatory LH are able to induce a significant loss of testicular LH/hCG binding capacity. Furthermore, after a short stimulatory phase there is a lack of response in testicular steroidogenesis to the elevated LH levels.


Physicochemical properties of the cytoplasmic triiodothyronine binding protein from tadpole liver and tail fin. The sedimentation velocity, gel filtration properties and pattern of elution from ion exchange gels of the cytoplasmic triiodothyronine (T3) binding protein from Rana catesbeiana liver and tail fin cytosol were determined. The T3 binding protein in liver cytosol had a sedimentation coefficient of 4.4S on sucrose gradients and a Stokes radius from Sephadex gel filtration of 38.2 +/- 4.2 A. From these two values a molecular weight of 71,700 +/- 4,100 and a frictional ratio of 1.28 +/- 0.05 for the T3 binding protein from tadpole liver has been calculated. The liver T3 binding protein was eluted from DEAE--Sephadex gels at a salt concentration of 0.233 +/- 0.017 M NaCl. The sedimentation coefficient, Stokes radius and salt requirement for elution from DEAE--Sephadex of the T3 binding protein from tail fin cytosol were essentially identical (4.4S, 35.2 +/- 4.2 A, nd 0.213 +/- 0.015 M NaCl respectively. The calculated molecular weight is 66,100 +/- 7,900 and the frictional ratio is 1.21 +/- 0.10 for for the tail fin T3 binding protein. The great similarity in the physicochemical properties of the T3 binding protein from the liver and tail fin implies that the T3 binding protein in each tissue is similar if not identical. The possible reason for the differences in the dissociation constants previously reported for the binding of T3 in the two tissues is discussed.


Lecithin: cholesterol acyltransferase activity in patients with chronic liver diseases and positive LP-X tests. Lecithin: cholesterol acyltransferase activity has been measured in serum from patients with various liver disorders and positive LP-X tests and from healthy subjects. Statistical analysis indicated that lecithin: cholesterol acyltransferase activity provided no help in the discrimination between the persons of both groups although the mean activities differed significantly.


Sensitivity difference of rat ascites hepatoma AH-13 and mouse leukemia L-1210 to nitrosourea derivatives. The structure-activity relationship in different sensitivity of AH-13 and L-1210 to nitrosourea nad related derivatives was examined. N-Methyl-N-nitrosoureido derivatives, such as 1-methyl-1-nitrosourea (MNU) and 1, 1'-polymethylene-bis(3-substituted 3-nitrosourea), were inactive against AH-13 and slightly active against L-1210. On the contrary, 1, 1'-polymethylene-bis(3-substituted 1-nitrourea) derivatives were more active against AH-13 than against L-1210. The nitrosoureas which had bis(2-chloroethyl) group at the terminal were highly active against AH-13 and L-1210. One of denitrosated derivatives, 1, 1'-ethylene-bis[3-(2-chloroethyl)urea], was active against AH-13 alone. In addition, diisocyanates, nitrourea, ammonium carbamate, and 1-methyl-1-nitrourea, which are related to the nitrosourea compounds, were also tested for their activity against AH-13 and L-1210. Diisocyanates and nitrourea were active against AH-13 alone, while other compounds were all inactive.


Detection of Epstein-Barr virus in biopsied malignant lymphoma cell and its continuous culture. In more than 80% of tumor cells in the pericardiac effusion of a case of malignant B-cell lymphoma, Epstein-Barr virus-determined nuclear antigen (EBNA) was detected by the anticomplement immunofluorescence test. Moreover, herpestype virus particles, although few in number, were demonstrated in the nucleus of lymphoma cells by an electron microscope. Tumor cells in the pericardiac effusion were seeded at 98% purity after centrifugation on Ficoll-Conray and, soon after plating, they proliferated continuously without any lag phase of growth or cell death. Therefore, the established cell line was regarded as of tumor cell origin and named Fujimaki-II cell after patient's name. On the other hand, Fujimaki-I cells were established from the biopsied tumor in the same way. These two cell lines, B-lymphocyte in nature, had both EB virus-related antigens and herpes-types virus particles. Heterotransplantation of cultured cells and tumor tissue obtained at autopsy into athymic nude mice was not successful. Transformation of cord blood lymphocytes by the virus released from Fujimaki-II cell also failed. This might be the first case of non-Burkitt type lymphoma in which the EB virus genome was directly detected in the tumor cells.


Mesotheliomas induced by sterigmatocystin in Wistar rats. Male Wistar rats were given repeated injections of sterigmatocystin (stg) and related compounds into the peritoneal cavity once a week for 23 weeks. Only stg alone produced mesothelioma in 20 of 40 rats given a total dose of 20 approximately 25 mg, while no mesotheliomas were found in rats injected O-acetyl-stg (AcO-stg) and dihydro-stg. There were two histological types in the mesothelioma induced by stg; the epithelioid and mesenchymal types. There was little loose connective tissue between the epithelioid cell tumors, whereas abundant dense collagen fibers were found between the cell nests of the mesenchymal cell types of mesothelioma. The hepatocellular carcinomas were found in a rat treated with stg and in five animals given AcO-stg. The fact that mesotheliomas could be induced in high incidence by direct intraperitoneal application of stg suggests a potent carcinogenicity of stg. Although both dihydro-stg and stg formed fine needle-like crystals when injected into the peritoneal cavity, dihydro-stg could not produce any mesothelioma. Dihydro-stg, reduction product of stg, has no double bond in the terminal furan ring. Therefore, the carcinogenicity of stg may be related more closely with the presence of a double bond in the terminal furan ring of stg than the physical form in the peritoneal cavity. AcO-stg may easily be absorbed from the peritoneum, so that it could not persist in the peritoneium for sufficient duration for carcinogenesis.


Lysosomal accumulation of gallium-67 in Morris hepatoma-7316A and Shionogi mammary carcinoma-115. Intracellular localization of gallium-67 was investigated in Morris hepatoma-7316A and Shionogi mammary carcinoma-115 cells by the cell fractionation method 48 hr after an intraperitoneal injection of the nuclide. When lysosomes were purified from both tumors by discontinuous sucrose density gradient centrifugation, they had a strikingly high relative specific activity of the nuclide. From these results it was confirmed that gallium-67 is concentrated most specifically in the lysosomes of both tumor cells, which consist chiefly of phagolysosomes and can engulf only limited amount of foreign materials such as Triton and gallium-67.


Motility of rat ascites hepatoma cells, with reference to malignant characteristics in cancer metastasis. The motility of four strains of rat ascites tumor cells in an agar medium was studied quantitatively by means of time lapse cinematography. The motility of these cells differed characteristically with each strain. Yoshida sarcoma and AH-66F cells showed active locomotion and partial movement markedly, while AH-130 and AH-100B cells made no locomotion and only a slight movement.


Comparison of carcinogenicity of N-alkyl-N'-nitro-N-nitroso-guanidines. Carcinogenicity of seven homologs of N-alkyl-N'-nitrosoguanidine (N-alkyl-NNG), namely, N-methyl-NNG, N-propyl-NNG, N-butyl-NNG, N-isobutyl-NNG, N-pentyl-NNG, and N-hexyl-NNG, was examined by injecting the compounds subcutaneously into rats. All of them except N-isobutyl-NNG and N-hexyl-NNG were carcinogenic, but N-ethyl-NNG followed by N-methyl-NNG were stronger carcinogens than those with longer alkyl chains.


Adenocarcinoma in tubular duplication of the sigmoid colon. A rare case of mucus-secreting adenocarcinoma in a tubular duplication of the sigmoid colon is presented. The pertinent radiographic manifestations were: a calcium containing soft tissue mass located at the blind end of a 20 cm barium filled tubular duplication originating at the sigmoid colon.


Benign hepatic lesions in women taking oral contraceptives. Four previously unreported cases of benign hepatic cell lesions in young women taking oral contraceptives are described. A brief review of the literature is presented with emphasis on the clinical symptoms, the findings on angiography and radionuclide scan, and the relation of the lesions to oral contraceptives.


Endotoxin and the liver. III. Modification of acute carbon tetrachloride injury by polymyxin b--an antiendotoxin. Previous work suggests that endotoxin of enteric origin may contribute to both acute and chronic liver injury by other agents. In particular, evidence exists that endotoxin tolerance modifies biochemical and histological evidence of carbon tetrachloride hepatotoxicity. The present study was undertaken to ascertain whether another method of modifying endotoxicity would protect against carbon tetrachloride (CCl4) damage as well. The antibiotic polymyxin B (PB) has unique antiendotoxin properties not shared by gentamicin sulfate, an antibiotic with a similar antibacterial spectrum. In groups of rats pretreated with either PB, gentamicin, or diluent, the LD100 of an oral dose of CCl4 was reduced by PB to an LD50, but the gentamicin pretreatment was without effect. When a sublethal dose of CCl4 was administered, both the SGOT and SGPT values were significantly lower in the PB group of rats. This biochemical protection was mirrored in the striking lack of histological liver necrosis in these animals, protection not shared by the gentamicin group or controls. The incidence of endotoxemia 24 hr after CCl4 as detected by lead acetate enhancement was also reduced by PB pretreatment. These findings further support the contention that endotoxins from the gut may be major contributors to the extent of liver injury induced by an unrelated toxin.


Augmentation of uridine diphosphate glucuronyltransferase activity in rat liver by adenosine 3',5'-monophosphate. The role of cyclic adenosine 3',5'-monophosphate (cAMP) in the regulation of rat liver bilirubin uridine diphosphate glucuronyltransferase (UDP-GT) was studied. Augmentation of UDP-GT activity was obtained by cAMP, but not by 3'-AMP. A single administration of glucagon initiated a rapid but limited increase in enzyme activity, which reached a maximum after 2 hr. Similar augmentation of the hepatic enzyme was produced by injection of N6,O2-dibutyryl cAMP. The nucleotide is the mediator for UDP-GT augmentation by glucagon. The injection of glucagon led within 20 min to a 40-fold increase in the concentration of cAMP. The augmentation of UDP-GT activity by glucagon or dibutyryl cAMP was fully inhibited by actinomycin D. A second stimulation of liver by glucagon or dibutyryl cAMP 4 hr after the first injection, produced a new increase of UDP-GT activity.


[Estrogen therapy and carcinoma of the endometrium (author's transl)]. The public discussion and the discussion among gynaecologists about the possible increased risk to develop carcinoma of the endometrium following post menopausal treatment with estrogens has lead to insecurity and restricted attitudes in the prescriptions of estrogens. In a statistic comparison of matched pairs of 130 cases of carcinoma of the endometrium with an equal number of selected control cases the following findings were elucidated: 1. Any estrogen preparations do not increase the relative risk for the development of carcinoma of the endometrium. 2. Differentiation into conjugated estrogens, estriol, oral contraceptives and various other estrogen compounds showed a similar trend. 3. Estrogen-androgen compounds and estrodiol compounds did not increase the relative risk for endometrial carcinoma. 4. The relative risk does not increase by prolonged ingestion of estrogen preparations in general and not for conjugated estrogens especially. 5. In the presence of other risk factors such as nulligravity, hypertension, obesity the relative risk for the development of carcinoma of the endometrium is significantly decreased when estrogens were taken, especially when the coincidental risk factor to estrogen ingestion was obesity. The above results suggest that the restraint in the prescription of estrogen for fear of endometrial carcinoma is not justified.


Prolyl hydroxylase activity in serum and rectal mucosa in inflammatory bowel disease. Prolyl hydroxylase activity in rectal mucosa was found to be significantly greater in 11 patients with Crohn's disease than in 11 control subjects with the irritable bowel syndrome and 16 patients with ulcerative colitis (P less than 0.005). Seven of the patients with Crohn's disease had a histologically normal rectum. This abnormality in apparently normal mucosa supports the concept that Crohn's disease is a 'continuous' disease of the gastrointestinal tract. Although there was no significant difference in prolyl hydroxylase activity between control subjects and patients with ulcerative colitis, those patients with quiescent disease tended to have lower values than those with active mucosal inflammation. Prolyl hydroxylase activity could not, however, be detected in the sera of either healthy control subjects or patients with inflammatory bowel disease.


[Encephalotomography and computer tomography for abnormalities of the sella (author's transl)]. Seventy-four patients with tumors in the sellar region were examined by means of encephalotomography and axial computerised tomography. In nearly all cases, the localisation and expansion of tumors could be exactly determined by encephalotomography. In addition, a graduation according to the degree of hypothalamic compression was possible. In cases of density differences between tumor and brain tissue and contrast enhancement more information of the inner structure of a tumor (cystic or solid) could be obtained by computerised tomography. But in cases of missing density differences or/and contrast enhancement tumor diagnosis is easier by encephalotomography because of the high efficiency of encephalotomography. The indication for the form of operative treatment, transsphenoidal or transcranial, depends on the exact knowledge of tumor growth, especially of the degree of hypothalamic compression. Therefore, in all uncertain computerised diagnosis encephalotomography has to be undertained.


[Internal carotid artery supply of juvenile angiofibromas (author's transl)]. Twelve juvenile angiofibromas of the paranasal sinuses are described which were partly supplied by branches of the internal carotid artery. Five vessels were found to be involved; with increasing frequency they were: the middle cerebral artery, the dorsal meningeal artery, the ethmoidal arteries, the artery of the inferior cavernous sinus and the artery of the pterigoid canal. Intracranial extension of tumour occurred only once, but the paranasal sinuses were involved in all cases. Occasionally, early filling of veins was seen. The branches of the internal carotid artery played a small part in the arterial supply when compared with branches of the external carotid artery. In spite of this, up to 80% of the tumour could be opacified through internal branches. Bilateral internal artery supply stresses the necessity for selective demonstration of both internal and both external carotid arteries.


Studies on the pituitary "Fettstoffwechselhormon". VIII. Radioimmunoassay of the lipolytic factor P-LF II D. A radioimmunoassay of the lipolytic peptide P-LF II D from porcine pituitaries is described. The assay is performed with 125-iodine labeled P-LF II D and with antisera either from guinea pigs or from rabbits. Bound antigen is separated from the free by double antibody technique. No cross reaction is observed with gamma lipotropin, peptide B, secretin, glucagon, isoproterenol. Due to contamination P-LF II C, beta lipotropin and human growth hormone displace the tracer when added at large doses. Complete cross reaction is observed between porcine 1-39 ACTH and P-LF II D. Specificity of this reaction is demonstrated by the increase of cross reaction, when ACTH fragments of increasing length of the polypeptide chain are used (1-23 ACTH, 1-24 ACTH and 1-28 ACTH).


Urinary cyclic AMP in hyperthyroidism.

Urinary cyclic AMP in hyperthyroidism. Urinary cyclic AMP was studied in 22 female and in 6 male hyperthyroid normocalcemic patients and in 3 hyperthyroid hypercalcemic men. Cyclic AMP/creatinine ratios were elevated both in female (4.12 +/- 0.26 mumoles/gm creatinine) and male (3.92 +/- 0.41 mumoles/gm creatinine) hyperthyroid normocalcemic patients as compared with normal female and male controls (2.85 +/- 0.20 and 2.54 +/- 0.14 mumoles/gm creatinine, respectively). However, there was no difference in the 24-hour urinary cyclic AMP excretion of both hyperthyroid and normal subjects. The hyperthyroid hypercalcemic men excreted less (2.47 +/- 0.19) mumoles/24 hr) cyclic AMP/24 hr than the normal male controls. In the thirteen female patients, studied when euthyroid, the cyclic AMP/creatinine ratio was normalised.


Cyclic AMP mediated increased amino acid transport: effect of isoproterenol administration. A single injection of isoproterenol (IPR) stimulates cell proliferation in rodent salivary glands after a lag period of about 24 hrs. Among the many events occurring prior to stimulated DNA synthesis, there is an early increase in cAMP levels and elevated transport of amino acids into the parotid gland. Amino acid transport is also elevated in liver and pancreas, tissues not induced to proliferate by IPR. IPR-stimulated alpha-aminoisobutyric acid (AIB) transport in parotid, pancreas and liver is augmented by prior injection of theophylline and is mimicked by dibutyryl cAMP. In all three tissues, changes in cAMP levels and subsequent increases in AIB transport appear to be closely related events. Since only the parotid gland is stimulated to grow after IPR injection, amino acid transport and growth would not appear to be directly related.


Iodide induced suppression of thyrotropin-stimulated adenosine 3',5'-monophosphate production in cat thyroid slices. Cat thyroid slices were studied to investigate their responsiveness to thyrotropin stimulation of cyclic AMP accumulation. Ovine and bovine thyrotropin, in the presence of 2.5 mM aminophylline, induced a dose-dependent increase in the cyclic AMP content of cat thyroid tissue. Half-maximal stimulation of cyclic AMP accumulation was obtained at a thyrotropin concentration of 1-2 mU/ml. The maximal effect of thyrotropin was observed at 10 mU/ml, and was associated with a mean 77 +/- 19-fold increase in thyroidal cyclic AMP. Preincubation of cat thyroid tissue for 2 h with 50 micron NaI resulted in an impairment in the subsequent ability of thyrotropin to enhance cyclic AMP accumulation, without altering the level of cyclic AMP in tissues not exposed to the hormone. Preincubation alone was without effect on thyrotropin stimulation of cyclic AMP, and the inhibitory effect of iodide was prevented by addition of 3 mM methimazole to the preincubation medium. In addition, the time course of thytrotropin stimulation of cyclic AMP accumulation in cat thyroid slices was not significantly altered by the preincubation with excess iodide. These studies provide additional evidence that excess iodide inhibits the adenylate cyclase-cyclic AMP system in thyroid tissue.


Monophasic synovial sarcoma--a histological entity? Consideration is given to the concept of a histologically identifiable monophasic type of synovial sarcoma. It is accepted that a sarcomatous tumour may be encountered where the spindle cells assume a somewhat epithelioid appearance associated with a reticulin pattern unlike that of most other spindle cell sarcomas. The appearance should alert a hsitopathologist to the possibility of a synovial sarcoma and prompt the examination of multiple additional sections in an attempt to find the pathognomonic biphasic pattern. It is not, however, believed that an entire tumour composed of cells of a single type could be identified with certainty as a synovial sarcoma by light microscopy. The term monophasic synovial sarcoma is worthy of retention, but only as a guide towards the establishment of a definite diagnosis by further sampling of the specimen. Other features suggestive of this diagnosis are discussed.


Primary carcinoma of the trachea.

Primary carcinoma of the trachea. Two cases of primary carcinoma of the trachea are described. Both patients were female and had smoked cigarettes for many years. One of the tumours was a squamous cell carcinoma and the other an oat cell carcinoma, the commonest and the rarest forms of tracheal cancer respectively. The diagnosis in the case of oat cell carcinoma was confirmed by electron-microscopic demonstration of neurosecretory granules, a unique observation at this site. Oat cell carcinoma of the trachea does not appear to have been recorded previously in a female.


The liver in coeliac disease.

The liver in coeliac disease. The pathology of the liver in 19 cases of malabsorption is reported. Five of these were proven to have adult coeliac disease, in the others that diagnosis was presumed by exclusion of other causes of malabsorption and by the coincidence of other conditions known to be associated with coeliac disease. Of these cases, three had liver changes of chronic hepatitis and two of these were in the proven coeliac group, including a case with cirrhosis and a hepatoma. In addition, less severe liver changes such as portal tract fibrosis and portal tract infiltration by inflammatory cells were present greatly in excess to that of the controls. The reasons for the occurrence of liver damage in coeliac disease are outlined and discussed in relation to the liver disorders associated with jejunoileal bypass used in the treatment of obesity. Possible mechanisms of liver injury in coeliac disease are described.


Histological discrimination of malignancy in mucinous ovarian tumours. Eight histological features were measured quantitatively in a group of 77 ovarian mucinous carcinomas and a group of 28 benign mucinous cystomas. These were compared with the duration of survival of the patients and the clinical staging of the tumours. Using the method of discriminant function analysis it was possible to identify a group of tumours of 'borderline malignancy'. Although single histological features were inadequate indicators of prognosis a combination of three features proved fairly accurate.


An improved syncytia infectivity assay for the bovine leukemia virus. Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 X 10(6) viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay.


The reduced extracellular calcium requirement for proliferation by neoplastic hepatocytes. Cells from neonatal rat livers were unable to maintain DNA-synthetic activity in calcium-deficient medium, but neoplastic hepatocytes from Morris hepatomas 5123 tc and 7795 synthesized DNA and proliferated indefinitely in this calcium-deficient medium. The calcium content of fresh hepatoma tissue from which these cultures were derived was as much as 10 times greater than that of normal liver; but this difference could not account for the insensitivity of neoplastic cells to extracellular calcium because it disappeared during subsequent cultivation in vitro.


On the relationship between inflammation and altered cAMP metabolism in lungs of B pertussis-vaccinated mice. Bordetella pertussis-vaccinated mice were examined for evidence of inflammation. Using polymorphonuclear leukocyte and fluid accumulation as markers, inflammation was evidenced in the lungs and to a lesser extent in the livers of such mice. Both heart and kidney showed no evidence of inflammation. Development of the inflammatory lesion followed a time course similar to that previously reported for increased sensitivity to histamine-mediated cAMP accumulation. This close parrallelism between inflammation and altered cAMP metabolism supports the hypothesis that the increased  cAMP accumulation might be related to a feedback mechanism regulating inflammatory mediator release.


Animal studies on the efficacy of an inactivated herpes simplex virus vaccine against recurrent herpes infection. The efficacy of inactivated herpes simplex virus vaccines for the treatment of recurrent herpes infection was studied in an animal model. Neither the frequency nor the severity of recurrent herpetic lesions could be influenced by long-term treatment of the animals with these vaccines.


Conditions required for induction of murine p30 by herpes simplex virus. Mouse cells (line N cIA cl10) contain 1.2-2.5 ng murine leukaemia virus (MuLV) p30 antigen/mg of protein; this amount of antigen is measurable by competition radioimmunoassay (RIA) but is not detectable by indirect immunofluorescence (IF). Infection of N cIA cl10 cells with herpes simplex virus type 2 (HSV-2) induces expression of MuLV p30. Induction by HSV-2 does not require either cell or virus DNA synthesis and is optimal 8 h post infection when cells at 50-70% confluence are infected at a multiplicity of infection (MOI) of 5-8 PFU/cell. At an MOI of 2.5, 70-80% of the cells express HSV antigens while none of the cells express p30; at an MOI of 5.0, 70-80% of the cells express HSV antigens but 55% of the cells express p30. Using the conditions reported in this paper for preparation of competing antigen, induction of p30 by HSV-2 (strain 333) infection is not measurable by competition RIA.


A portion of the feline leukaemia virus genome is not endogenous in cat cells. Although viral sequences closely related to feline leukaemia virus are represented in multiple copies in cellular DNA of all domestic cats, a specific fraction was present only in the virus-infected cells. This fraction was detected by viral cDNA enriched by a prior absorption of a total complementary DNA (cDNA) transcript with normal cat liver DNA. The recycled cDNA hybridized well with the cellular DNA of virus-infected cells, but to a lesser extent with DNA from uninfected cat cells. The probe was used to differentiate virus-positive from virus-negative tumour tissues of cats. The same approach with cDNA of another endogenous feline virus, RD114, failed to show any difference between a virus-infected cell line and normal cells, including both virus-inducible and non-inducible lines.


Enzymatic digestion of DNA gamma-irradiated in aqueous solution separation of the digests by ion-exchange chromatography. Aqueous solutions of DNA were gamma-irradiated in the presence and absence of oxygen and enzymatically hydrolysed by the combined action of pancreatic deoxyribonuclease (DNase I), snake-venom phosphodiesterase (PDE I), spleen phosphodiesterase (PDE II) and alkaline phosphatase. In contrast to unirradiated DNA, which is fully hydrolysed to nucleosides by these enzymes, gamma-irradiated DNA yields a series of oligonucleotides. Their isolation might enalbe the future identification of the chemical nature of DNA lesions.


Inhibition of x-ray-induced protection of Escherichia coli K-12 cells against the lethal effects of ultra-violet light by nitrofurantoin. Wild-type cells of E. coli K-12 showed increasing U.V. resistance if they were X-irradiated and incubated at 37 degrees C in growth medium before the U.V. exposure. Development of higher U.V. resistance could be inhibited by incubating the X-irradiated cells either at temperatures below 15 degrees C, or in the presence of 0.01 M KCN. Nitrofurantoin (NF), which was recently found specifically to inhibit inducible enzyme synthesis, had only a transient inhibitory effect on X-ray-induced U.V. resistance. Cells grown in glucose medium showed less inhibition by NF of X-radiation-induced resistance to U.V.-radiation than did cells grown in glycerol, or in glucose medium with added cyclic AMP. It is suggested that X-ray-induced U.V. resistance requires active cellular metabolism, but it is not subject to catabolite repression. The following hypothesis is offered to explain the action of NF: Under de-repressed conditions (without catabolite repression by glucose) nitrofurantoin could counteract the radiation-induced inhibition of a repair inhibitor (such as post-irradiation DNA degradation).


Antigen-inducing ability of herpesvirus papio in human and baboon lymphoma lines, compared to Epstein-Barr virus. Herpesvirus papio(HVP)-carrying baboon lymphoblastoid lines do not express a nuclear antigen like the Epstein-Barr virus(EBV)-determined nuclear antigen (EBNA), as judged by in situ anticomplement fluorescence staining, although the carry multiple viral genomes and, in the case of producerlines, early antigen (EA) and viral capsid antigen (VCA) that cross-react with the corresponding human EBV-determined antigens. To test whether the lack of in situ nuclear antigen expression is a property innate to the baboon virus or the baboon cell, nonproducer HVP-carrying baboon lymphoid cells of the 26 CB-1 line were superinfected with two human EBV strains. B95-8-derived EBV induced brilliant EBNA staining, proving that the baboon lymphoid cell was competent to synthesize EBNA. In the mirror experiment, HVP derived from the 9B or the 18C baboon line was added to the EBV-carrying Raji line, the EBV-negative Ramos and BJAB lines and the HVP-carrying nonproducer 26 CB-1 line, respectively. HVP induced EA and VCA in Raji, and EA in BJAB and 26 CB-1. EBNA was not induced in any of the three EBNA-negative lines, BJAB, Ramos and 26 CB-1. It is concluded that the lack of in situ nuclear staining in HVP-carrying baboon lines is a HVP-associated property and is not due to any innate inability of the baboon lymphoid cell to synthesize an antigen of the EBNA type.


Temperature-sensitive mutants of simian virus 40. I. Isolation aand preliminary characterization of B/C gene mutants. Sixteen temperature-sensitive mutants of simian virus 40 were isolated after treatment of wild-type virus with UV light, nitrous acid, or nitrosoguanidine. All of the mutants were assigned to the B complementation group on the basis of quantitative complementation analyses. Characterization of several representative mutants revealed that under restrictive conditions such mutants produced immunologically identifiable tumor and viral capsid antigens, induced the synthesis of cellular DNA, and replicated infectious viral DNA. In addition, mutant particles produced at 33 degrees were more heat-labile than wild-type virus. This collection of mutants will be useful in an analysis of SV40 particle morphogenesis.


Differential inhibition of host cell RNA synthesis in several picornavirus-infected cell lines. Patterns of cellular RNA synthesis during poliovirus (types 1,2 and 3) and mengovirus infections in HeLa, HEp-2, Vero and L-cells were investigated by means of high-resolution autoradiography, kinetics of 3H-uridine incorporation, and sucrose gradient centrifugation. Only mengovirus-infected L-cells showed an early and exponential inhibition of hnRNA and rRNA synthesis. The other cell lines tested, whether infected with polio- or mengovirus, exhibited a slowly and linearly decreasing cellular RNA synthesis, with hnRNA production suppressed completely, although somewhat later than in L-cells, and rRNA synthesis continuing at a gradually reduced rate during the entire viral growth cycle. Locations of cleavage steps of rRNA precursor molecules are discussed.


Synthesis and packaging of herpes simplex virus DNA in the course of virus passages at high multiplicity. Herpes simplex virus type 1 (HSV-1), strain ANG, was passaged serially at high MOI or undiluted on RC-37 cells. The yields of infectious virus decreased and increased periodically with a maximum of about 3 log units variation in titer. The amount of newly synthesized HSV-1 DNA, however, did not vary by more than a factor of 2. On the other hand, the fraction of total HSV-1 DNA associated with virions was reduced at passage numbers coinciding with the minima in the yields of infectious progeny virus. Apparently, low yields of infectious virus mainly reflected a reduced efficiency of packaging of viral DNA into mature virus particles. One step in the process of virus maturation which appeared to be impaired was the assembly of nucleocapsids. HSV-1 ang was shown to be capable of generating different classes of defective particle in independent series of virus passages at high MOI.


Effect of caffeine on the replication of nonirradiated and ultraviolet-irradiated cytomegalovirus. The effect of caffeine on cytomegalovirus (CMV) replication in human embryo lung (HEL) and human embryo kidney (HEK) cells was studied. In HEL cultures, nonirradiated CMV yielded up to 6.7 times more plaques in caffeine-treated cultures than in control cultures. Virus growth curves exhibited different profiles, depending on the time of treatment of the cultures with the drug. Incorporation of [3H]TdR into virus DNA was enhanced and began about 24 h earlier in the caffeine-treated cultures than in the untreated cultures. A relationship between decreased incorporation of [3D]TdR into cellular DNA caused by caffeine and increased virus replication was observed in the HEL cells. In caffeine-treated HEK cultures, CMV replicated better than in untreated cultures in which virus growth was transitory and decreased after 10 days of incubation. The infectivity of CMV irradiated with UV light doses of 3,120-12,480 erg/mm2 decreased as a function of the UV dose. The extent of multiplicity reactivation of the irradiated virus increased when irradiated with higher UV doses. The effect of caffeine on the infectivity of UV-irradiated CMV depended on the dose with which the virus was irradiated. Caffeine increased the infectivity of CMV irradiated with lowest UV dose (3,120 erg/mm2) but did not increase the infectivity of virus irradiated with 12,480 erg/mm2. Multiplicity reactivation of the UV-irradiated CMV was inhibited by caffeine regardless of the UV dose with which the virus was irradiated.


Transmission of B77 virus double mutant LA334 markers by transfection. Successful transfection of Brown Leghorn chicken fibroblasts was carried out with DNA isolated from duck cells transformed by the LA334 mutant of avian sarcoma virus B77. Transfection of duck cells was negative. The four viruses recovered after transfection were all temperature-sensitive for transformation. Two were fully temperature-sensitive for replication, as shown by analysis of virus replication, by characterization of virus particles produced at the nonpermissive temperature using density gradient centrifugation, and by electron microscopic examination. The other two viruses were only partially temperature-sensitive for replication. The results suggest that both the src and gag regions of the avian sarcoma virus genome are transferred simultaneously during transfection, probably by a single integral provirus copy.


Nuclear accumulation of filamentous herpes simplex virus DNA late during the replicative cycle. New ultrastructural findings within the nucleus of herpes simplex virus-infected cells are illustrated. The occurrence of bundles of tightly packed filaments during the late stages of the infectious process is described. These bundles were found in different areas of the nucleus and were not associated with any nuclear organelles. Employing different staining techniques and high-resolution autoradiography, they could be identified as DNA-containing nucleoproteins. These filaments may be interpreted as a special form of viral DNA accumulation within the nucleus. Changes of these structures toward a more recticular arrangement were observed as soon as the nucleoplasm disintegrated.


Purification of hepatitis A virus from human feces. Hepatitis A virus was purified fecal specimens obtained from 2 patients with naturally acquired hepatitis A. The purification procedure involved differential centrifugation, organic solvent extraction, agarose gel filtration, ion-exchange chromatography, and isopycnic ultracentrifugation in cesium chloride. Using immune electron microscopy and discontinuous SDS-PAGE, this procedure was found to be effective in removing extraneous material from hepatitis A virus. There was significant recovery of virus as judged by immune electron microscopy and solid-phase radioimmunoassay. Using this protocol, it has been possible to obtain virus preparations of sufficient purity and high enough titer to enable biochemical studies to proceed.


Herpesvirus morphology: visualization of a structural subunit. The presence of spontaneously disrupted cytomegalovirus capsids has made it possible to visualize a subunit within the herpesvirus capsomere. The pattern resolved agreed well with the theoretical net based on the concept of a structural subunit at each apex of the hexon.


Seroepidemiologic aspects of varicella zoster virus infections in an Israeli Jewish population. Sera from 872 healthy subjects among the Jewish population of Israel were examined for antibodies to varicella zoster virus (VZV) by a simplified technique for the detection of immunofluorescent antibody to membrane antigen. The difference in the geometric mean titer of antibodies to the virus among the the different age groups was highly significant (P less than 0.0001). The highest titers were found in the five-to-seven-years and 21-to-30-years age groups. In 211 subjects, aged 17 to 40 years, the antibody titres were not influenced by sex or ethnic origin. The percentage of seronegative subjects (titer less than 2) was relatively high--between 18.7 and 32.9% in the 21-to-60-years age groups--and it was 19.5% in the population of childbearing age. The importance of screening for VZV immune status in pregnant women is emphasized.


Population distribution of histological types of lung cancer: epidemiologic aspects in Israel and review of the literature. A nationwide study of lung cancer in Israel during 1968-70 demonstrated a low to moderate incidence in men and a high incidence in women, as compared with other countries. The sex ratio was relatively lower than expected. This discrepancy results from a low rate of squamous cell carcinoma in men and a high rate of adenocarcinoma in women, and is consistent with similar, previously observed trends in North American Jews from New York, Pittsburgh and Montreal. The literature relating to the relative frequencies of various histological subcategories of lung cancer is reviewed and the importance, for etiology and prognosis, of a separate assessment of each distinct cell-type entity is emphasized.


Chemical and electrophoretic changes induced by polymyxin B on outer membrane components from Serratia marcescens. The effects of polymyxin B (PB) on outer membrane (OM) components from resistant (strain 08) and sensitive (strain Bizio) cells of Serratia marcescens were characterized by chemical analysis and polyacrylamide gel electrophoresis (PGE) in sodium dodecylsulfate. Chemical analysis revealed no major differences in the OM fractions after PB treatment of both strains, except for the loss of protein in PB treated OM of the sensitive strain. The yield and composition between the lipopolysaccharides (LPS) of the two strains were different both before and after treatment. PGE revealed that there was a complex formation between the LPS of resistant strain and PB but both dissociation and degradation occurred in the LPS components in the sensitive strain. In addition, it was found that the protein components were destabilized by PB with subsequent loss of some of the components from OM of the sensitive strain. The difference in the amount of LPS and their ability to complex with PB may reflect different degrees of antibiotic susceptibility of these two strains of S. marcescens. A sequential multistep mechanism is proposed for the action of PB on outer membranes of this species.


Method for maintaining viability of Clostridium perfringens in foods during shipment and storage: collaborative study. A collaborative study was conducted in 12 laboratories to determine the effectiveness of a new method for maintaining vegetative cells of Clostridium perfringens in viable condition during storage and transport of food specimens to the laboratory. The collaborative results showed that treatment of brown gravy and roast beef samples with an equal amount by weight of sterile buffered glycerol-sodium chloride solution to give a final 10% glycerol concentration and storage with Dry Ice for 10 days at -56 degrees C resulted in plate counts of C. perfringens which were 2-4 log cycles higher with 2 different strains than counts with untreated specimens stored by the usual method at -20 degrees C. Plate counts obtained with the treated specimens stored with Dry Ice were less than 1 log cycle lower than counts made with identical specimens before freezing for storage and shipment to the collaborators. The results with treated specimens were also more uniform among the different laboratories. Because the new method for storage and shipment of food samples was so effective for maintaining viability of the organism, the official first action method for C. perfringens (46.B01) was changed to incorporate these procedures as part of the method.


Galactose catabolism in Caulobacter crescentus. Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.


Induction of collagenase production in Vibrio B-30. The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.


A mutation altering some properties of the neutral phosphatase in Chlamydomonas reinhardi: possible post-translational modification of phosphatase structure. A mutant (PDs-) of Chlamydomonas reinhardi has been isolated which produces an altered neutral phosphatase. The wild-type (PDs+) and mutant (PDs-) phosphatases markedly differed in their thermosensitivities and electrophoretic mobilities. The heterozygous PDs-/PDs+ diploids produced only the wild-type electrophoretic form of the phosphatase. Mixing extracts of PDs- with extracts of various other strains in vitro resulted in the rapid transformation of the PDs- enzymic form into an enzymic variety, the properties (heat sensitivity, electrophoretic mobility) of which were similar to those of the wild-type neutral phosphatase. The results are discussed in relation to the idea that the PDs mutation is located not in the structural gene but rather in a modifying gene acting at the post-translational level.


Expression of the histidine operon in rho mutants of Escherichia coli. Expression of the Escherichia coli histidine operon was measured in four independently isolated sets of strains carrying ten different rho mutations. Rho factor does not act as a major regulatory element of histidine operon attenuation.


Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724. Immunochemical studies demonstrated that Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 produces only a single diol dehydratase whether grown on glycerol or on 1,2-propanediol. The enzyme was subject to induction by 1,2-diols and to catabolite repression reversed by cyclic AMP.


Enzyme-accelerated hydrolysis of polyglycolic acid. In a preliminary study of the enzyme-polymer interactions, the role of 15 enzymes in the in vitro hydrolysis of polyglycolic acid has been investigated. Carboxypeptidase A, alpha-chymotrypsin, clostridiopeptidase A and ficin increase the rate of hydrolysis of this synthetic polymer, illustrating the ability of enzymes to influence polymer degradation.


The interaction of liver alcohol dehydrogenase with phenyl adenine dinucleotide, a novel analog of pyridine nucleotide coenzymes. We have synthesized phenyl adenine dinucleotide (P1-adenosine-5')-P2-(beta-D-ribofuranosylbenzene-5')-pyrophosphate (PhAD), a novel analog of pyridine nucleotide coenzymes. This compound contains a planar aromatic ring, as does NAD+, but lacks a positive charge. PhAD is an inhibitor of horse liver alcohol dehydrogenase, competitive with NADH. PhAD is very similar to NAD+ sterically since both compounds have a planar aromatic ring. However, PhAD resembles NADH in binding to the enzyme because the dissociation constants of both compounds show a parallel increase as the pH is raised, whereas those of NAD+ behave in the opposite manner. These observations indicate that the enzyme differentiates between NAD+ and NADH on the basis of the positive charge on the molecule and not the stereochemical orientation of the reduced nicotinamide ring.


Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase. Cytoplasmic fructose-1,6-bisphosphatase has been purified from spinach leaves to apparent homogeneity. The enzyme is a tetramer of molecular weight about 130,000. At pH 7.5, the Km for fructose 1.6-bisphosphate was 2.5 micron, and for MgCl2 0.13 mM; the enzyme was specific for fructose 1,6-bisphosphate. Saturation with Mg2+ was achieved with lower concentrations at pH 8 than at pH 7. AMP and high concentrations of fructose 1,6-bisphosphate inhibited enzyme activity. Ammonium sulfate relieved the latter inhibition but was itself inhibitory when substrate concentrations were low. Acetylation studies demonstrated that the AMP regulatory site was distinct from the catalytic site. Cytoplasmic fructose-1,6-bisphosphatase may contribute to the regulation of sucrose biosynthesis in plant leaves.


Hepatic alpha-adrenergic receptors. Identification and subcellular localization using [3H]dihydroergocryptine. Recently, several workers have shown that adrenergic control of hepatic carbohydrate metabolism has the characteristics of an alpha-receptor-mediated process. Using the rat liver membrane preparation of Neville (Neville, D. (1968) Biochim. Biophys. Acta 154, 540-552), alpha-adrenergic receptors have been identified using the ligand [3H]dihydroergocryptine. The receptors are saturable and of high affinity. Scatchard analysis yields a KD of 1.8 nM with 1.7 +/- 0.55 pmol of sites/mg of protein. Competition of dihydroergocryptine binding with various pharmacologic agents yields the typical (alpha-adrenergic potency series: (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol. (-)-Isomers are more potent than (+)-isomers. The alpha-blocker phentolamine is 3.4 orders of magnitude more potent than the beta-blocker propranolol. To determine subcellular localization of alpha-adrenergic receptors, livers were fractionated into a crude homogenate, a 1500 X g pellet, and the purified membrane preparation used previously for binding. Specific dihydroergocryptine binding, ouabain-inhibitable (Na,K)-ATPase, and F--stimulated adenylate cyclase activities, were followed in these fractions. Specific binding was enriched, relative to that in the crude homogenate, 2.88-fold in the pellet and 6.28-fold in the membranes. Similarly, (Na,K)-ATPase acticity was enriched 2.6-fold in the pellet and 7.1-fold in the membranes while adenylate cyclase activity was enriched 2.9-fold in the pellet and 3.5-fold in the membranes. It is concluded that hepatic alpha-adrenergic receptors are likely concentrated in the plasma membranes.


Conversion of glucose to sorbitol and fructose by liver-derived cells in culture. Conversion of glucose to fructose and sorbitol is documented in rat hepatoma-derived cultured cells (HTC cells). After addition of 5.5 mM [U-14C]glucose to incubation medium, labeled sorbitol and fructose accumulated intracellularly at a linear rate over a period of 60 min. The sugars were isolated, identified, and quantitated by paper chromatography, gas-liquid chromatography, and enzymatic phosphorylation of fructose. Primary culture of adult rat hepatocytes was analyzed similarly and demonstrated no significant accumulation of labeled fructose or sorbitol. The basis for this difference between HTC cells and primary hepatocyte culture was examined both in terms of enzyme activities that mediate the formation of sorbitol and fructose and in terms of the catabolism of these sugars. Both types of culture (as well as extracts of intact rat liver) exhibited enzymatic activities catalyzing the conversion of glucose to sorbitol (aldose reductase) and sorbitol to fructose (sorbitol dehydrogenase). However, the cultures differed strikingly with regard to the catabolism of sorbitol and fructose. The conversion of labeled sorbitol to metabolites in HTC cells was negligible; by contrast, hepatocytes in primary culture utilized the sugars at rates comparable to that of glucose, which may account for the lack of their accumulation in primary culture. The findings suggest that the conversion of glucose to sorbitol and fructose by HTC cells may represent a retained normal liver function, one which is amplified by the inability of HTC cells to dispose of these sugars.


5-Methylthioribose kinase. A new enzyme involved in the formation of methionine from 5-methylthioribose. The presence of a previously unidentified enzyme, tentatively designated 5-methylthioribose kinase, has been demonstrated in cell-free extracts of Enterobacter aerogenes. The enzyme catalyzes the ATP-dependent phosphorylation of 5-methylthioribose. ADP is one of the products of the reaction and, based on functional group analyses, the other product is 5-methylthioribose 1-phosphate. A 40-fold purified enzyme preparation has been obtained from a cell-free extract of E. aerogenes. Activity of the partially purified enzyme is totally dependent on the presence of a divalent cation and a sulfhydryl reagent. The substrate specificity of the enzyme is quite narrow, and the Km values for ATP and 5-methylthioribose are 7.4 X 10(-5) M and 8.1 X 10(-6) M, respectively. These results suggest that 5-methylthioribose kinase may be a primary enzyme involved in the recycling of the methylthio group of 5-methylthioribose back into methionine.


Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus-transformed hamster fibroblasts. The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells grown in medium containing [14C]- or D-[3H]glucosamine have been separated into two distinct classes: a phenol-soluble fraction and an aqueous fraction. The membrane glycoproteins from both BHK21/C13 and C13/B4 partitioned similarly into these two fractions. The phenol and aquesous-soluble glycoproteins differed in their sodium dodecyl sulfate-polyacrylamide gel profiles, polyacrylamide isoelectric focusing profiles, and glycopeptide distribution on Sephadex G-50. A number of aqueous and phenol-soluble glycoproteins from BHK21/C13 and C13/B4 cells were purified to near homogeneity by means of polyacrylamide electrophoresis and gel electrofocusing. These glycoproteins range in molecular weight from 179,000 to 31,000 and have isoelectric points of 7.5 to 3.0. Our results show that the pronase glycopeptides of 20 out of 24 homologous membrane glycoproteins of equivalent molecular weight and isoelectric point from BHK21/C13 and C13/B4 cells are dissimilar as measured by Sephadex G-50 gel filtration.


Adenosine 3':5'-monophosphate receptor proteins in mammalian brain. cAMP receptor proteins present in mammalian brain were identified and characterized with the use of the photoaffinity label 8-azido[32P]cAMP. Cytosol and membrane fractions from various regions of rat, guinea pig, and bovine brain contained two specific cAMP receptor proteins with apparent molecular weights of 47,000 and 52,000 to 55,000. Subcellular fractionation studies showed the highest amounts of these cAMP receptor proteins associated with cytosol fractions and synaptic membrane fractions. For both the cytosol and membrane fractions, the two cAMP receptor proteins represented almost all of the proteins specifically labeled by 8-azidol[32P]cAMP and appeared to be the regulatory subunits of cAMP-dependent protein kinases.


The use of orthacryl two-dimensional polyacrylamide gel electrophoresis to identify and compare the subunit polypeptides of bovine heart and yeast cytochrome c oxidases. A two-dimensional electrophoretic method which takes advantage of the "migration anomalies" experienced by some polypeptides on gels of different porosities has been successfully used to resolve the seven subunit polypeptides of yeast cytochrome c oxidase and the nine polypeptides associated with bovine cytochrome c oxidase. The two-dimensional maps provided by this method reveal clear differences between these two cytochrome c oxidases.


Interaction of 125I-labeled Ca2+-dependent regulator protein with cyclic nucleotide phosphodiesterase and its inhibitory protein. The Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase was labeled with 125I to the extent of 1 mol of monoiodotyrosine per mol. The iodinated protein showed a small decrease in affinity for phosphodiesterase but gave the same maximal level of activation of the enzyme as did the unmodified regulator protein. Iodinated regulator protein formed complexes with both highly purified cyclic nucleotide phosphodiesterase and phosphodiesterase inhibitory protein in the presence but not in the absence of Ca2+ as demonstrated by ultracentrifugation in glycerol gradients. Cross-linking experiments indicate that the Ca2+-dependent regulator protein interacts with the large subunit of the inhibitory protein.


The K+/site and H+/site stoichiometry of mitochondrial electron transport. Electrode measurements of the average number of H+ ejected and K+ taken up (in the presence of valinomycin) per pair of electrons passing the energy-conserving sites of the respiratory chain of rat liver and rat heart mitochondria have given identical values of the H+/site and 5+/site ratios very close to 4 in the presence of N-ethylmaleimide, an inhibitor of interfering respiration-coupled uptake of H+ + H2PO4-. The K+/site uptake ratio of 4 not only shows that inward movement of K+ provides quantitative charge-compensation for the 4 H+ ejected, but also confirms that 4 charges are separated per pair of electrons per site. When N-ethylmaleimide is omitted, the H+/site ejection ratio is depressed, because of the interfering secondary uptake of H/+ with H2PO4- on the phosphate carrier, but the K+/site uptake ratio remains at 4.0. Addition of phosphate or acetate, which can carry H+ into respiring mitochondria, further depresses the H+/site ratio, but does not affect the K+/site ratio, which remains at 4.0. These and other considerations thus confirm our earlier stoichiometric measurements that the average H+/site ratio is 4.0 and also show that the K+/site uptake ratio can be used as a measure of the intrinsic H+/site ratio, regardless of the presence of phosphate in the medium and without the necessity of adding N-ethylmaleimide or other inhibitors of H+ + H2PO4- transport.


A reassessment of structure-function relationships in glucagon. Glucagon1-21 is a full agonist. Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (less than 0.001 residue/mol) and the activity of the compound did not change after treatment with cyanogen bromide as has been shown with native glucagon. Glucagon1-21 stimulates hepatic adenylate cyclase activity to the same extent as native glucagon but with 0.1% the potency. Glucagon1-21 also displayed 0.1% the binding affinity of native glucagon to the glucagon receptor in hepatic membranes. Glucagon22-29 alone or in combination with glucagon1-21 did not activate adenylate cyclase or displase 125I-glucagon from its receptor. The finding that glucagon1-21 is a full agonist on adenylate cyclase is discussed in relation to the structure-function relationships required for the biological action of glucagon.


Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. Adenylate cyclase can be resolved into at least two proteins, a thermolabile, N-ethylmaleimide-sensitive component and a second protein (or proteins) that is more stable to either of these treatments. Neither component by itself catalyzes the formation of cyclic AMP using MgATP as substrate. However, mixture of the two reconstitutes MgATP-dependent fluoride- and guanyl-5'-yl imidodiphosphate (Gpp(NH)p)-stimulatable adenylate cyclase activity. The more stable component can be resolved from the first in various tissues or cultured cells by treatment of membrnes or detergent extracts with heat or N-ethylmaleimide. The two proteins have also been resolved genetically in two clonal cell lines that are deficient in adenylate cyclase activity. An adenylate cyclase-deficient variant of the S49 lymphoma cell (AC-) contains only the thermolabile activity, while the activity of the more stable protein is found in a complementary hepatoma cell line (HC-1). In addition, AC-S49 cell plasma membranes contain MnATP-dependent adenylate cyclase activity. The protein that catalyzes this reaction appears to be the same as that which can combine with the thermostable component to reconstitute Mg2+-dependent enzyme activity because both activities co-fractionate by gel exclusion chromatography and sucrose density  gradient centrifugation, both activities have identical denaturation kinetics at 30 degrees C, and both activities are stabilized at 30 degrees C and labilized at 0 degree C by various nucleotides and divalent cations with similar specificity. It is thus hypothesized that the thermolabile factor is the catalytic subunit of the physiological adenylate cyclase and that the Mn2+-dependent activity is a nonphysiological expression of the catalytic protein. The thermostable moiety of the enzyme, which is proposed to serve a regulatory function, appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity. These components have not, however, been physically separated. The thermolabile and thermostable components can interact in detergent solution or in a suitable membrane. Mixing of the detergent-solubilized regulatory component with AC-membranes that contain only the catalytic protein and beta-adrenergic receptors reconstitutes catecholamine-stimulatable adenylate cyclase activity; however, addition of the catalytic protein to membranes that contain receptor and the regulatory component yields MgATP-dependent enzymatic activity that is unresponsive to hormone.


Characterization of functional receptors for somatostatin in rat pituitary cells in culture. GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a  hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.