Identification of a sperm receptor on the surface of the eggs of the sea urchin Arbacia punctulata. The possibility that the surface of the egg of the sea urchin Arbacia punctulata contains a species-specific receptor for sperm has been investigated. The extent of fertilization of eggs of A. punctulata, which is proportional to the number of sperm, is unaffected by the presence of either eggs or membranes prepared from eggs of Strongylocentrotus purpuratus. In marked contrast, membranes prepared from eggs of A. punctulata quantitatively inhibit fertilization of A. punctulata eggs by A. punctulata sperm. Several lines of evidence indicate that this inhibition is due to the presence of a membrane-associated glycoprotein that binds to the sperm, thus preventing them from interacting with receptor on the surface of the eggs. First, eggs treated with trypsin are incapable of being fertilized, although they can be activated with the Ca2+ ionophore A23187. Moreover, membranes prepared from eggs pretreated with trypsin do not inhibit fertilization of eggs. Second, receptor isolated in soluble form from surface membranes binds to sperm and thus prevents them from fertilizing eggs; the inhibition by soluble receptor is species-specific. Third, the soluble receptor binds to concanavalin A-Sepharose. Fourth, eggs are incapable of being fertilized if they are pretreated with concanavalin A. The specificity of inhibition, and the affect of trypsin and concanavalin A on intact eggs, suggest that the receptor is a species-specific macromolecule located on the surface of the eggs. The sensitivity of the receptor to trypsin, and its ability to bind to concanavalin A, indicate that it is a glycoprotein.


Do aging changes in the preoptic area contribute to loss of cyclic endocrine function? Two-year-old Wistar rats showing repeated periods of pseudopregnancy were compared with young rats that had electrolytic lesions placed in the medial preoptic area. Electrolytic lesions in the medial preoptic area in young rats induced repeated pseudopregnancies what were similar to those seen in the old pseudopregnant rats. Serum prolactin levels were elevated in both groups of rats. Administration of lergotrile mesylate (an ergot-dervied dopamine agonist and prolactin inhibitor) resulted in a reduction in serum prolactin levels and in the appearance of normal consecutive 4- or 5-day estrous cycles in the old rats and in the young rats with brain lesions. These results suggest that the repeated periods of pseudopregnancy in the aged rats may result from a deficiency in dopamine or from an abnormality in dopamine metabolism in neurons arising or passing through the medial preoptic area.


Mepacrine, a tool for investigating the 5-hydroxytryptamine organelles of blood platelets by fluorescence microscopy. In the blood platelets of various species exposed to mepacrine, the average number of green-yellow fluorescent granules (probably identical with the 5-hydroxytryptamine [5-HT] storage organelles) corresponded to that of flashes emitted by the platelets on prolonged irradiation with violet-blue light. In platelets of fawn-hooded rats the number  of granules did not markedly differ from that of normal rat platelets, but the fluorescence intensity and the uptake of mepacrine in vitro showed a marked decrease and the flashes were less numerous. The heavy population of human platelets exhibited considerably more granular structures than the light population. The data suggest that (1) in normal, mepacrine-loaded platelets one flash corresponds to one 5-HT organelle and (2) mepacrine is a useful tool for investigating the number and function of the 5-HT organelles in live platelets and possibly for studying platelet age.


Hypotonic exchange-loading of erythrocytes. II. introduction of hemoglobins S and C into normal red cells. A rapid, simple technique whereby erythrocytes transiently exposed to hypotonic hemoglobin preparations undergo exchange of endogenous for exogenous hemoglobin has now been applied to normal, hemoglobin A-containing erythrocytes into which hemoglobin S or hemoglobin C have been introduced. Erythrocytes containing an average of 12 to 61 percent of hemoglobin S undergo sickling when incubated in isotonic dithionite solution and are morphologically indistinguishable from natural sickle cells. Ultrastructural studies of thin sections of these artificial sickle cells show microfialments and fascicles which appear typical of microtubular tactoids of hemoglobin S seen in natural sickle cells. Erthrocytes containing 43 percent introduced hemoglobin C developed all of the morphologic alterations associated with intra-erythrocytic hemoglobin C, including hemoglobin aggregation, targetting, and intracellular crystallization, after inclbation for 24 hours in isotonic saline: dithionite. The results support the concept that the sickling process, and morphologic erythrocyte alterations occurring in hemoglobin C disorders are primary to the respective hemoglobins rather than the membrane. They also suggest that any role played by the inner surface of the erythrocyte membrane in the development of these alterations is probably secondary and not specific to the inner membrane surface of the natural sickle cell or hemoglobin C-containing cell.


Trigeminal artery and microemboli to the brain stem. Report of two cases. The authors report two cases of transient ischemic attacks (TIA's) involving the brain stem. The TIA's were due to microemboli that originated from a carotid bifurcation atherosclerotic plaque and travelled through a persistent trigeminal artery.


Fate of small diameter cervical veins grafted into the common carotid arteries of growing rabbits. Autogenous cervical veins were grafted into the common carotid arteries of rabbits during their active growing period. A patency rate of 58.8% was achieved but the thin-walled vein grafts underwent massive dilatation. This dilatation is likely to severely limit the usefulness of these veins as a source of bridging grafts in clinical practice, particularly for use within the intracranial cavity.


Cranial zygomycosis caused by Saksenaea vasiformis. Case report. A previously healthy youth who had sustained severe head trauma and had received steroids and broad-spectrum antimicrobial agents developed a cranial zygomycotic infection with Saksenaea vasiformis. This is the first time this zygomycete has been implicated as a disease agent. Early identification of the fungal infection and subsequent vigorous medical and surgical therapy led to recovery.


Dynamic imaging of the systemic perfusion of a lung cancer: case report. Dynamic lung circulation scintigraphy employing intravenously administered 99mTc as pertechnetate was used to visualize the pulmonary arterial and systemic arterial circulations in a patient with bronchogenic carcinoma. Computer-processed dynamic images and region-of-interest data were utilized to demonstrate differences between tumor and normal regions regarding pulmonary and systemic blood supplies.


Principles of management of the persistent cloaca in the female newborn. The persistent cloaca in the female newborn is one of the most complex and challenging developmental malformations that the pediatric urologist will encounter. A review of 5 patients seen at this center and 34 completely documented cases was undertaken to characterize the defects and devise a logical diagnostic and therapeutic approach. Diagnostic steps include the search for associated anomalies, which were present in the upper urinary tract (33 per cent), gastrointestinal tract (13 per cent), cardiovascular system (13 per cent), central nervous system (10 per cent) and respiratory tract (5 per cent). Delineation of the cloaca and its anatomic relationships is determined by abdominal x-rays, injection of contrast material into the cloacal channel, excretory urography and endoscopy with selective catheterization. Surgical treatment begins with a diverting right transverse colostomy in the newborn period followed by rectal pull-through when the patient weighs 25 pounds. Vaginal pull-through should be performed at the same time as the rectal pull-through if the vagina enters the cloaca high. If the cloacovaginal communication is low a vaginoplasty by a posterior flap procedure can be done when the child is 10 to 12 years old. Even is anatomy is reconstructed in an ideal manner urinary and fecal continence cannot be assured since congenital neurologic dysfunction is not uncommon.


Long-term experience with porcine aortic valve xenografts. Between 1971 and 1975, glutaraldehyde-preserved porcine aortic valve xenografts were employed for isolated replacement of the mitral valve (MVR) in 243 patients, replacement of the aortic valve (AVR) in 167 patients, and double valve replacement (AVR and MVR) in 51 patients. Postoperatively, long-term anticoagulation was not routinely given. Operative mortality rates for AVR, MVR, and double valve groups were 7.8, 6.0, and 11.8 per cent, respectively; the majority of early postoperative deaths were associated with concomitant coronary artery disease. No death was attributable to xenograft dysfunction. Follow-up of all patients was obtained. The total duration of follow-up for the MVR group was 347 patient-years, for the AVR GROUP 148 148 patient-years, and for double valve replacement 37 patient-years; maximum follow-up for these three groups was 4.4, 4.0, and 2.4 years, respectively. Actuarial analysis of postoperative survival rates at a common interval of 3 years showed 78 per cent for MVR patients, 91 per cent for AVR patients, and 80 per cent (projected) for patients with double valve replacement (85, 96, and 91 per cent for operative survivors, respectively. At this same interval 92 per cent of MVR patients, 99 per cent of AVR patients, and 93 per cent (projected) of patients with double valve replacement were free of thromboembolic episodes. Altogether, 12 of the total 512 valves implanted exhibited some evidence of dysfunction during the entire period of follow-up evaluation, but in only 2 instances (both mitral) was intrinsic pathological involvement of the xenograft tissue documented. Actuarial analysis of xenograft dysfunction at a common interval of 3 years after operation showed 95 per cent of MVR patients, 98 per cent of AVR patients, and 97 per cent (projected) of patients with double valve replacement to be free of this complication. These data support the use of glutaraldehyde-preserved porcine xenografts as superior bioprostheses that pose a low risk of thromboembolism without anticoagulation. The over-all durability of such valves, within the restriction of a maximum current follow-up interval of 4.4 years, appears comparable to that of currently available mechanical prostheses and justifies continued clinical use.


Isometric cardiac contraction. a possible cause of the disorganized myocardial pattern of idiopathic hypertrophic subaortic stenosis. The bizarre septal architecture of idiopathic hypertrophic subaortic stenosis (IHSS) may be secondary to a small systolic cavity with late systolic isometric contraction. We examined ventricular muscle for IHSS-type muscle-fiber disarray in infant hearts in which isometric contraction would occur during development-namely, pulmonary or aortic-valve atresia with intact ventricular septum and normal atrio-ventricular valves. Fifteen patients with aortic atresia and 10 with pulmonary atresia were compared to 25 normal controls of matched age and heart weight. Aortic atresia showed disorganization of muscle-fiber alignment of left ventricle, particularly septum, and intramural coronary-artery changes virtually identical to IHSS. Pulmonic atresia had similar right ventricular  disarray and vessel changes, again most marked in the septum. Thus, cardiac muscle-cell disorientation similar to IHSS occurs in infant ventricles with outflow-tract obstruction. This IHSS-type myocardial-fiber disarray may result from altered wall stresses related to isometric systolic contraction.


Fetal movements and placental lactogen levels for fetal-placental evaluation. A preliminary report. Fetal movements were measured by 37 pregnant women during a 10-minute period while they were lying on their left side at various times during the day with the highest rates in the evening. Preliminary assessments of fetal movement and serum hPL levels in pregnant women suggest that the two tests might complement each other in providing more information about the status of the placenta and fetus.


Guidelines for the elective induction of labor with oral Prostaglandin E2. Fifty multiparous patients at 37 weeks or more of gestation with a vertex presentation and a Bishop score of 7 or more had labor induced with oral Prostaglandin E2 tablets. All responded and delivered vaginally. The induction to delivery time averaged 4 hours and 44 minutes. The average number of tablets required was 3.64. Four women experienced nausea and an additional 4 women had some vomiting. Two patients showed hypertonus and 11 had frequent contractions. Many progressed rapidly after they began active labor. There was no evidence of fetal distress. Guidelines are suggested for the use of oral Prostaglandin E2 tablets to adequately control the labor process and prevent hypercontractility.


Galactorrhea and amenorrhea in a patient with an empty sella. A report of a patient with amenorrhea and galactorrhea who was shown, by tomopneumoencephalogram, to have an empty sella is presented. Endocrinologic testing revealed only a blunted human growth hormone response to insulin-induced hypoglycemia and acyclicity of plasma gonadotropins. Thyroid testing, ACTH, and metopirone responses were normal. In addition, plasma prolactin levels were found to be within the normal range. Most significantly, after the pneumoencephalogram the patient's menses returned and have continued at regular monthly intervals for 6 months. During this time there has been a significant decrease in the galactorrhea. This is the first patient described with an empty sella and galactorrhea. The clinical and endocribologic aspects of the empty sella are discussed.


Cutaneous stimulation and generation of breathing in the fetus. The generation of spontaneous regular breathing by cutaneous stimulation and by direct electrical stimulation of the sciatic nerve was examined in six previously apneic mature fetal lambs in utero. The fetuses were stable throughout the course of the experiments: PaO2 less than or equal to 27 mm Hg, PaCO2 less than 44 mm Hg, pH 7.29-7.34, blood pressure and heart rate steady and normal. It is shown that electrical stimulation of the fetal skin (66 cps, 4.0 msec, 6 V, 0.77 ma) can be as effective as direct stimulation of the sciatic nerve (66 cps, 4.0 msec, 1.5 V, 0.08 ma) when the higher voltage and current are used. Mechanical cutaneous stimulation also produced spontaneous breathing which, however, was short lived compared with that produced by electrical stimuli. The results are consonant with our concept of activation and recruitment of quiescent respiratory center neurones by somatic sensory stimulation, and they give fundamental support to the clinical observation of others that cutaneous stimulation is effective for the treatment of apnea of prematurity. Speculation Somatic sensory stimuli from the skin may be important determinants of the onset of breathing in the fetus and newborn.


Imbalance of globin chain synthesis in newborn infants with hemolytic disease after exchange transfusion. The relative rates of beta-, gamma-, and alpha-globin chain formation in reticulocytes of six newborn infants ,ith hemolytic disease were determined before and in weekly intervals after exchange transfusion. Directly after birth (before exchange transfusion) the beta/alpha, gamma/alpha, and (beta + gamma)/alpha ratios in newborn infants with hemolytic disease (0.35 +/- 0.20, 0.67 +/- 0.12, and 1.06 +/- 0.29) were similar to those of normal newborn infants (0.33 +/- 0.07, 0.65 +/- 0.06, and 0.98 +/- 0.06). The gradual increase in the beta/alpha ratio seen during the postnatal period of eight normal newborn infants was also observed in newborn infants with hemolytic disease after exchange transfusion. In contrast, the gamma/alpha ratio was found to be increased (1.89 +/- 0.25) in all infants with hemolytic disease in the second week after exchange transfusion. The corresponding value for normal newborn infants was 0.48 +/- 0.25. After exchange transfusion the number of hemoglobin F-containing erythrocytes increased. Possible reasohs for this imbalance of globin chain formation after exchange transfusion may be the decreased erythropoiesis and/or the abnormal high hemoglobin A concentration after exchange transfusion or the introduction of an unknown, gamma chain formation-stimulating agent by exchange transfusion. Speculation In newborn infants with hemolytic disease the repression of gamma-globin chain formation normally seen during the postnatal period is replaced by a stimulated gamma-globin chain production after exchange transfusion.


Lateral transport of a lipid probe and labeled proteins on a cell membrane. Diffusion coefficients (D) of a lipid probe and labeled proteins on L-6 myoblast membranes have been measured giving D(protein) approximately 2 X 10(-10) square centimeter per second and D (lipid probe) approximately 9 X 10(-9) square centimeter per second. Some of the membrane proteins are immobile, but the lipid probe diffuses freely over macroscopic distances. Cytochalasin B slows protein but not lipid probe diffusion.


Properties of bovine fibrin absorbable implants. The assimilation characteristics of absorbable bovine fibrin implants in an experimental situation are described, and the effect of delaying absorption with formaldehyde treatment is demonstrated. Essentially, in vivo in the rat, Biethium fibrin implants are absorbed in approximately four to eight weeks because of the polymorphonuclear leukocyte digestion. An indication of the clinical uses in stress incontinence and hemorrhage of the liver are given, together with a look at the future of some of the other areas in which this absorbable, biologic plastic may fit into the surgeon's armamentarium to replace deficient or destroyed tissue.


Clinical use of immunoelectrophoresis of split ejaculates. I. Variation of patterns due to antisera. As a preliminary investigation for the clinical application of immunoelectrophoresis of ejaculate in the diagnosis of male reproductive organ diseases, variations of immunoelectrophoretic patterns in normal subjects due to the difference of antigenicity between split ejaculates were demonstrated with rabbit antisera. Fresh samples and antisera against them gave larger numbers of and more intense precipitin lines than in stored samples. The precipitin lines developed by acid phosphatase were most remarkable between first portion of ejaculate (F1) and anti-F1, whereas the line caused by protein from the seminal vesicle was most prominent between the remainder of the ejaculate following F1 (F2) and anti-F2.


Integral storage and voiding reflexes. Neurophysiologic concept of continence and micturition. It is a common clinical misconception to regard the spinal micturition reflex center as fundamentally overactive and dependent on cerebral inhibition. Initiation and cessation of micturition is simplistically viewed as a manifestation of voluntary withdrawal and resumption of inhibitory corticospinal "regulation''. This view is in conflict with basic neurophysiologic experimental data. Actually, the organization of the micturition reflex is extremely complex. It is affected by multiple sources of facilitative and inhibitory influence, peripheral as well as central. During the past half century, at least twelve reflexes involved in urine storage and coordinated micturition have been described by various neurologic investigators. In this article the integral reflexes are identified and described. A functional organization of the integral reflexes which includes a modern concept of their role in the physiology of urine storage and micturition is presented. It is implicit that overactivity or functional failure of any one or combination of the integral reflexes may cause a significant disorder of lower urinary tract function.


Dog bites.

Dog bites. Dog bites can be prevented by proper education of children, appropriate selection of family pets and the restraint of guard dogs in the community. An unusually high incidence of bites by German shepherds was observed in this study. Treatment of injuries inflicted by dogs should include: careful wound toilet, appropriate excision of damaged tissues, careful wound repair with adequate drainage and systematic therapy with selected antibiotics.


Complete heart block as a cause of syncope in asymmetric septal hypertrophy. This case report presents a young adult with asymmetric septal hypertrophy (ASH) and syncope. Infranodal complete heart block was demonstrated as his cause for syncope. Therapy consisted of implantation of a A-V sequential pacemaker. Cardiac output determinations and systolic time intervals demonstrated the beneficial effects of properly timed atrial contractions.


Development of "subaortic stenosis" after pulmonary arterial banding for common ventricle. Progressive narrowing of the bulboventricular foramen is documented in four patients with single ventricle. The morphologic features in each case corresponded to single left ventricle with infundibular chamber. Two patients had a D-ventricular loop and the other two an L-loop. All four patients had transposition of the great arteries. Restriction of the bulboventricular foramen thus resulted in functional subaortic stenosis because the aorta originated above the infundibular chamber. The development of subaortic obstruction was documented by serial cardiac catheterization studies in two infants, one of whom underwent unsuccessful surgical enlargement of the bulboventricular foramen. In the other two patients the initial cardiac catheterization revealed no pressure gradient between the ventricle and aorta, but examination at necropsy revealed very severe obstruction at the bulboventricular foramen, thus suggesting that the obstruction had been acquired. In each patient, the progressive narrowing of the bulboventricular foramen occurred after pulmonary arterial banding and may have been causally related to this procedure. This functional subaortic obstruction developed in 4 of 31 patients (12.9 percent) with single left ventricle, transposition of the great arteries and pulmonary arterial banding. Clinical recognition of this development is predicated on (1) awareness that narrowing of the bulboventricular foramen in patients with single ventricle and pulmonary arterial banding may be common; (2) presence of symptoms such as angina; and (3) lack of continued clinical improvement in a patient whose pulmonary arterial band has significantly reduced pulmonary blood flow. Management of this subaortic stenosis requires surgical intervention. In the infant, a ventriculotomy in the outlet chamber will usually provide excellent exposure of the bulboventricular foramen, and surgical enlargement can be accomplished. In the older child with severe obstruction, marked muscle hypertrophy may obliterate the ventricular cavity, making ventricular partitioning difficult if not impossible.


Thrombotic thrombocytopenic purpura with C'3 vascular deposits: report of a case. A case of thrombotic thrombocytopenic purpura in a young woman in which platelet microthrombi were distributed in small vessels throughout the body is reported. IgM and complement (C'3) were demonstrated in capillary and small arteriole walls by immunofluorescent microscopy. These findings support the findings of a previous case report, and are further evidence for a possible primary immune etiology for thrombotic thrombocytopenic purpura.


Glucose-6-phosphate dehydrogenase isoenzymes in acinar epithelial cells separated from the mammary gland of the rat at intervals during the course of lactation. Acinar epithelial cells were purifed from suspensions of cells from the lactating mammary glands of rats. As described previously, this purififaction was accomplished by sedimentation in an isokinetic gradient of Ficoll (polysucrose) in tissue culture medium, and the purity of the separated cells was established by electron microscopy and by histochemical markers. Isoenzymes of glucose-6-phosphate dehydrogenase were investigated at various intervals during lactation in separated populations of stromal and acinar cells. Acinar cells contained three- to eightfold more glucose-6-phosphate dehydrogenase activity than did stromal cells. The proportions of the respective isoenzymes varied during the course of lactation, and the observed changes were parallel in purified acinar cells and in the lactating mammary glands from which the cell suspensions were obtained. The availability of purified acinar cells in the investigation of interactions between hormones and cells from the mammary gland permits a greater degree of specificity than has been possible in the study of mammary cell suspensions which contain myoepithelial cells, duct cells, acinar cells, endothelial cells, fibroblasts, fat cells, mast cells, plasma cells, and blood cells.


Assessment of vagal control of the heart in diabetes. Measures of R-R interval variation under different conditions. R-R intervals and R-R interval variation were measured in 2 selected groups of diabetic subjects; 1 group was selected because vagal control of the heart appeared normal, the other group was selected because they lacked vagal control of the heart. The means of the R-R intervals for the 2 groups were not significantly different under various conditions (sleeping, lying, during deep breathing, sitting, standing, and during upright activity). The standard deviations of the R-R intervals for the 2 groups were only significantly different during deep breathing and upright activity. It is suggested that cardiac vagal function is not reliably assessed by measurement of R-R interval variation during quiet standing, but useful information may be obtained during deep breathing.


End-diastolic amplitude of mitral valve echogram in mitral stenosis. By using simultaneous recordings of the mitral valve echogram and apex cardiogram, the mitral echogram amplitude was measured at the onset of left ventricular isovolumic contraction (MAIC). Twenty normal subjects and 68 patients with a reduced diastolic closure rate in the mitral valve echogram were studied. Of these patients, 53 had mitral stenosis, 6 aortic valvar stenosis, and 9 hypertrophic obstructive cardiomyopathy. In the normal subjects the MAIC ranged between 2 and 4 mm, average 2-7 mm, in the patients with aortic valvar stenosis or hypertrophic obstructive cardiomyopathy between 2 and 4 mm, average 2-9 mm, and in the patients with mitral stenosis between 6 and 17 mm, average 11-3 mm. The DE/MAIC ratio, where DE represents the opening amplitude of the mitral valve in early diastole, was between 3-3 and 6-5, average 5-1, in normal subjects; in the patients with aortic stenosis or hypertrophic obstructive cardiomyopathy the DE/MAIC ratio was between 2-7 and 6-5, average 4-2, and in the patients with mitral stenosis between 0-7 and 1-5, average 1-1. An excellent correlation was found between the DE/MAIC ratio and mitral valve area in the patients with mitral stenosis (r = 0-84, P less than 0-01) while the correlation between the diastolic closure rate and valve area was less satisfactory (4 = 0-62, P less than 0-01). These findings suggest that in cases with a reduced diastolic closure rate for reasons other than mitral stenosis, error can be avoided by using the DE/MAIC ratio.


Structural basis of heme reactivity in myoglobin and leghemoglobin: thermal difference spectra. Thermal pertubation difference spectra of sperm whale myoglobin (Mb) and soybean leghemoglobin a (Lb a) in the near-ultraviolet reveal similarities in the tryptophan environment of the two proteins. Of the two tryptophans in each protein, one has its indolyl NH group fully exposed to aqueous solvent, while the other behaves as if it were surrounded by motile but nonpolar residues with little access to water. These environments are not significantly altered by removal of the heme group. Assuming conformational homology, the helix-spacing role of Trp-A12 in Mb (Kendrew, J.C. (1962), Brookhaven Symp. Biol. 15, 216-228) may be taken over, in Lb a, by Trp-H8 which, though remote in linear sequence, would occupy a suitable spatial location. Thermal difference spectra in the Soret and visible regions of pure high-spin (fluoroferric) and pure low-spin (cyanoferric) complexes showed a red shift on cooling Mb complexes, reflecting a predominantly nonpolar environment around the heme, but a blue shift on cooling Lb complexes, reflecting a more solvent-exposed environment. Thermal difference spectra using rose bengal as a probe of the heme pockets in the two apoproteins supported these conclusions. Thermal difference spectra for the high-spin complexes of both Mb and Lb are slightly larger in magnitude than in the low-spin complexes. This may reflect a more flexible heme pocket in the high-spin state, as suggested by recent circular dichroic results. A structural basis for the high oxygen affinity of Lb compared with Mb is proposed, based upon the observed differences in polarity and flexibility of the heme pocket and in amino acid substitutions.


Interaction of palladium (II) with DNA.

Interaction of palladium (II) with DNA. The nature of interaction of palladium (II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.


Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos. Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.


Computerized tomography using the EMI general purpose scanner. The EMI general Purpose Scanner is briefly described, with particular reference to the use of window height and window width variations. Patient preparation in relation to movement artefacts and contrast medium is considered, and a method of localization of tomographic sections is described. The Scanner produces brain examinations of high quality. Because a head water bath is no longer required, the base of the skull can also be examined. In the thorax computerized tomography is particularly valuable in showing pleural disease in the forms of thickening, plaques or tumours, and in demonstrating retrosternal lymph node enlargement. Small peripheral metastases can be identified when not shown by conventional tomography. In the abdomen lymph node masses, retroperitoneal tumours and pancreatic disease has been identified, often more completely than by other methods, or where other methods have failed. This has proved particularly valuable in the grading of malignant disease and in the monitoring of subsequent treatment. It is particularly helpful in the planning of radiotherapy. Metastases in liver and bone can be clearly defined when still quite small (0-5.1 cm), but comparison with other methods of diagnosis such as grey scale ultrasound and isotope scanning will be needed before the role of computerized tomography in evaluating these lesions is known.


Single unit recording in the midbrain of rats during shock-elicited fighting behavior. Single unit activity was recorded extracellularly from the midbrain of rats during fighting behavior and during non-fighting control manipulations. Fighting behavior was elicited by footshock or startle stimuli or occurred spontaneously as a result of prior footshock presentations. Seven cells were found in the midbrain reticular formation and central gray which displayed maximum firing rates during fighting behavior. These cells also fired to a limited extent to some of the control manipulations, particularly contralateral vibrissae stimulation. These cells fired phasically during fighting behavior and their firing was correlated with either the approach or paw-strike of the opponent animal or to the response of the recording animal to a tactic of the opponent animal. However, no specific movement or sensory event reliably predicted the firing of these cells during fight sequences. Cells located in other midbrain areas, such as the deep tectum or the area of the red nucleus, also responded during fighting behavior. However, the discharge of these cells was correlated with specific body movements or sensory events. The activity during fighting was similar in rate and pattern to activity during control manipulations whenever similar movements or sensory stimulation were produced. Cells were also found which did not discharge during fighting behavior although they fired under a variety of other conditions.


Parallel changes in behaviour and hippocampal serotonin metabolism in rats following treatment with desglycinamide lysine vasopressin. Application of a foot shock during the acquisition trial of a one-trial passive avoidance task is associated with a rise in the concentration of serotonin in the hippocampus 24 h after conclusion of the acquisition trial. Carbon dioxide (CO2) induces amnesia for the passive avoidance response when administered immediately upon termination of the acquisition trial. In rats subjected to CO2 treatment following foot shock the rise in hippocampal serotonin is not observed 24 h later. The vasopressin analogue desglycinamide lysine vasopressin attenuates CO2-induced amnesia for the passive avoidance response when given prior to either the acquisition or the retrieval test (24 h after acquisition). This attenuation of the passive avoidance response is associated with a rise in the hippocampal serotonin concentration similar to the one observed in non-amnesic animals. It is suggested that a correlation exists between changes in hippocampal serotonin metabolism and the retrievability of the passive avoidance response.


The evaluation of tests.

The evaluation of tests. In order to correctly evaluate a test, at least four attributes should be measured: namely, sensitivity, specificity, accuracy and precision. Sensitivity is the proportion of diseased animals which are correctly identified, whereas specificity is the proportion of healthy animals which are correctly identified. These two attributes are important, not only because of the reasons implied by their definition but because they influence both the apparent prevalence of disease and proportion of test-positive animals which are actually diseased. The ability of a test to give a true measurement of the substance being measured, its accuracy, and its ability to give consistent results on the same sample, its precision, are good measures of quality control. Both these attributes influence the sensitivity and specificity of the test. Inaccuracies and inconsistencies arise from the test itself, the technician and the nature of the sample being tested.


Effect of hydrolyzed formaldehyde-treated RNA on neoplastic and normal human cells. Alkaline hydrolysate of dialyzed formaldehyde-treated RNA, in which 39% of purine nucleotides are modified to methylene bis derivatives (R-CH2-R'), displays cytotoxic activity toward cultured human tumor cells. The alkaline hydrolysate of dialyzed formaldehyde-treated RNA is substantially less toxic to normal human embryonic cells. There is a correlation between the cytotoxic effect and cell growth rate, which has been traced by quantitative tests.


Culture of cloned cells from the mouse preputial gland tumor ESR 586. Cells from the mouse preputial gland tumor ESR 586 have been cultured and cloned. Trypsin-ethylenediaminetetraacetate was used to obtain single cells. The cells are grown in a modified CMRL-1415 medium supplemented with 10% fetal calf serum. Clones tend to fall into two classes: Class 1, those that undergo morphological differentiation in liquid medium to form round bodies filled with lipid vaculoes; and Class 2, those that do not differentiate. Preliminary studies on the control of differentiation in Class 1 clones suggest that a minimum cell density is required before differentiation takes place. Analysis of the lipids from differentiating and nondifferentiating clones reveals the presence of sebaceous-type lipids in differentiating clones only. No requirement for testosterone was found for these cells.


Comparison of echocardiographic and necropsy measurements of ventricular wall thicknesses in patients with and without disproportionate septal thickening. In several patients with asymmetric septal hypertrophy (ASH) diagnosed by echocardiography (septal-free wall thickness ratios greater than or equal to 1.3), we have discovered marked discrepancies between the echocardiographic and necropsy measurements of wall thicknesses that led to uncertainty regarding the actual cardiac diagnosis. To resolve these apparent incongruities, the echocardiograms and hearts of 17 patients with cardiac disease were studied. Six of nine patients with abnormal septal-free wall ratios greater than or equal to 1.3 during life had septal-free wall ratios that were not diagnostic of disproportionate septal thickening at necropsy. Such discrepancies may be explained as follows: 1) echocardiographic measurements during life were made in diastole (as per convention), but measurements at necropsy were made in hearts that appeared to have been in the systolic phase of the cardiac cycle; 2) the left ventricular free wall thickens considerably more than the ventricular septum in systole, as determined by echocardiography. This latter phenomenon resulted in septal-free wall ratios in systole that were consistently smaller than those in diastole. Furthermore, septal-free wall ratios obtained at necropsy corresponded most closely to those obtained by echocardiography in systole.


Complete heart block in a case of idiopathic hypertrophic subaortic stenosis: noninvasive correlates with the timing of atrial systole. A young man with IHSS who developed complete heart block was successfully treated with a permanent pacemaker. Echocardiography and other noninvasive techniques showed marked cycle-to-cycle variation in the evidence of subvalvular obstruction which decreased markedly when atrial systole preceded the ensuing paced complex by an appropriate interval. Because cycle length and therefore afterload were constant, it is concluded that diminished obstruction resulted from augmented ventricular end-diastolic volume produced by atrial contraction. The mitral valve echocardiogram showed unusual movements in diastole dependent upon the timing of atrial systole. Early reopening of the leaflets was a direct result of atrial contraction when the P waves were appropriately timed in presystole, whereas late reopening was passive and a result of ventricular filling in mid-diastole. Variations in intensity of the first heart sound correlated with the position of the mitral valve leaflets at the onset of ventricular systole.


A radioimmunoassay for human serum myoglobin: method development and normal values. We describe a radioimmunoassay that will measure both normal and above-normal concentrations of myoglobin in serum. Myoglobin isolated from human pectoralis muscle was purified by (NH4)2SO4 fractionation and Sephadex gel filtration and injected into rabbits to elicit antisera. Myoglobin was radiolabeled by an acylation with [125]-3-(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester. With the purified myoglobin and antisera, we then developed a radioimmunoassy that involves simultaneous regent addition, a 3.5-h incubation at 37 degrees C, and separation of the antibody-bound fraction by precipitation with polyethylene glycol. Information is given on detection limit, precision, linearity, recovery, and specimen preservation. Cross-reactivity to human hemoglobin is negligible. Finally, we investigated the possible relationship between serum myoglobin concentration and muscle mass.


Purification and iodination of antibody for use in an immunoradiometric assay for serum ferritin. Antibodies were purified by affinity chromatography and radioiodinated by a conjugation method for use inthe immunoradiometric assay for serum ferritin. These procedures are simpler and more reproducible than those described earlier, and the radiolabeled preparations were usable for at least 13 weeks.


Disseminated intravascular coagulation complicating aflatoxicosis in dogs. An outbreak of chronic liver disease was investigated in a kennel of dogs. Anorexia, depression, polyuria, polydipsia, icterus and a terminal hemorrhagic diathesis were noted in clinically affected dogs. Thrombocytopenia, hypofibrinogenemia, elevated fibrinogen degradation products and prolonged activated partial thrombosplastin times (PTT) and one-stage prothrombin times (PT) were associated with the hemorrhagic crisis. Aflatoxicosis was confirmed by the presence of significant levels of aflatoxicosis was confirmed by the presence of significant levels of aflatoxin B in the commercial dog food being fed. A subacute hepatitis was found on necropsy. Disseminated intravascular coagulation was suspected as the cause of the hemorrhage in these cases and treatment was instituted.


Sex steroids in follicular fluid and blood plasma during the estrous cycle of pigs. Concentrations of estrogen (E), progestin (P),and androgen (A) were determined in follicular fluid (FF), and concentrations of E and P were measured in peripheral plasma and perifollicular plasma during the estrous cycle in the pig. Also, concentrations of E, P,and A were determined in the utero-ovarian vein (U-OV) plasma every 2h during estrus period in pigs. It was observed that as follicles increased in size, the concentration of E, P, andA in the FF rises from 13,32 and 1 ng/ml at day 8 of the cycle to 180, 754, and 21 ng/ml at day 20 of the cycle, respectively. Approximately 12 h before ovulation there is a significant (P less than 0.01) drop of E, P, and A in the FF to 27, 120, and 0.77 ng/ml, respectivley. Concentrations of E and P in FF and peripheral plasma do not correlate during the second half of the estrous cycle. At day 20 of the cycle, E and P concentrations in the plasma were 20 pg/ml and 0.4 ng/ml, respectively. Concentration of P in the U-OV plasma started increasing 18 to 24 h after the onset of estrus, which is coincident with the drop of P concentration in the FF. As estrus progressed there was a linear drop of E concentration (r = 0.90) in the U-OV plasma. E and P concentrations in the FF were found to be about 1000 times greater than those in the peripheral blood and 5 times greater than in the blood obtained from the perifollicular capillary network at the end of the cycle.


Coitus-induced release of luteinizing hormone in the proestrous rat: fantasy or fact? The influences of mating on serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were evaluated in 4-day cyclic proestrous rats of the Wistar strain. Blood samples were collected via guillotine from mated and unmated controls at 1900-2000 h or 2100--2200 h. Serum LH levels were determined by two independent radioimmunoassays. There were no significant differences in LH measurement between assays. At 1900--2000 h, LH levels were high, but not significantly different, in both the mated and unmated control animals. By 2100-2200 h, serum LH concentration had dropped in unmated controls but was significantly elevated in mated animals. There was no significant elevation in FSH either 1900-2000 h or 2100--2200 h on the evening of proestrus. The data indicate that when LH levels are normally high, coitus cannot further potentiate LH release. However, when LH levels are declining, coitus is an effective and adequate stimulus to initiate elevations in serum LH.


Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy. Catecholamine levels in hypophysial portal plasma were determined in pregnant and non-pregnant female rats as well as in intact and castrated male rats, using a radioenzymatic assay for the simultaneous determination of dopamine, norepinephrine, and epinephrine in 50 mul of plasma. Portal and arterial blood were collected from anesthetized rats at 7 mul/min for 60 min. During the collection, blood was kept at 0 C, a temperature at which endogenous catecholamines were relatively stable. Dopamine was present in high concentrations in hypophysial portal plasma thorughout pregnancy, attaining a level near 20 ng/ml on the 20th day of gestation. Dopamine levels in arterial plasma from the same rats were low or undetectable (0.4--0.8 ng/ml1. Norepinephrine and epinephrine was undetectable (less than 0.6 ng/ml) in portal as well as arterial plasma from these rats. The major catecholamine in extracts of the hypothalamus from pregnant rats was norepinephrine, whereas that in the posterior pituitary was dopamine. Dopamine levels in portal plasma collected during proestrus, estrus, diestrus 1, and diestrus 2, were 1.32 +/- 0.21 (mean +/- SE), 3.87 +/- 0.96, 3.11 +/- 0.73, and 2.3 +/- 0.45, respectively. Dopamine in portal plasma from intact and from castrated male rats was approximately 0.6 ng/ml. Norepinephrine and epinephrine were not detectable in either portal or arterial plasma from these animals. It is concluded 1) that dopamine is secreted into hypophysial portal blood in significant quantities during pregnancy, 2) that hypothalamic secretion of dopamine in cyclic rats is greatest during the day of estrus and early diestrus and at least on the day of proestrus, and 3) that these findings support the view that dopamine of hypothalamic origin may have an important role in the regulation of anterior pituitary function.


Opiate-like action of methionine-enkephalin in inhibiting morphine abstinence syndrome. Intracerebroventricular administration of methionine-enkephalin or morphine sulfate immediately prior to naloxone administration inhibited the precipitated withdrawal jumping response in mice rendered dependent on morphine by the pellet implantation method. Both methionine-enkephalin and morphine sulfate failed to inhibit withdrawal defecation and rearing behavior. Morphine sulfate was found to be four times as potent as methionine-enkephalin, on molar basis, in inhibiting the abstinence syndrome. These data provide new in vivo pharmacologic evidence for the opiate-like action of methionine-enkephalin.


Potentiating effect of lithium chloride on methamphetamine-induced stereotypy in mice. Single administration of LiCl potentiated the methamphetamine-induced stereotypy in mice, without affecting the concentration of methamphetamine in brain. During MAO inhibition, the level of 3-methoxytyramine (3-MT), an O-methylated metabolite of dopamine, was reduced by LiCl, but the elevated 3-MT level observed after the administration of methamphetamine was not influenced by LiCl.


Adhesion and aggregation of human platelets to rabbit subendothelium. A new approach for investigation: specific antibodies. An SgG antibody occurring in a recently transfused thrombasthenic patient inhibited all the ADP-mediated aggregations and platelet-platelet interaction (thrombus formation) on rabbit aorta subendothelium; another IgG antibody occurring in a multitransfused Bernard-Soulier patient inhibited ristocetin and bovine factor VIII mediated aggregation and platelet-subendothelium interaction.


Human cervical mucus. I. Rheologic characteristics. The viscoelastic properties of human cervical mucus were subjected to detailed characterization by microrheometry. An introduced to the range of parameters that can be measured and the sensitivity of microroheometry to variations in mucus viscoelasticity was presented, along with an interpretation of results obtained by this approach. Fresh cervical mucus samples were shown to be rheologically heterogeneous, reflecting compositional differences in situ. Mucas viscoelasticity was stable for several hours when samples were stored at ambient temperature in the microrheometer sample holder. Mucus viscoelasticity was strongly dependent upon nondialyzable solids (NDS) concentration. In order to eliminate (NDS) as a variable in comparative studies of mucus, procedures were developed for reconstitution of mucus at identical (NDS) or for normalization of viscoelasticity data derived from fresh samples to 2.5% NDS. The validity of this approach was then examined by applying these procedures to mucus collected during the follicular, ovulatory, and luteal phases of the normal menstrual cycle.


Influence of the estrous cycle on the action of oxytocin on rat ovarian contractility in vivo. This investigation was designed to determine the influence of the estrous cycle on the response of ovarian smooth muscle to oxytocin. Muscular activity in vivo was studied by recording intraovarian pressure. Intra-aortic injections of oxytocin were given to the rats at all stages of the estrous cycle. The effect of oxytocin was evaluated by the number of responding ovaries and the magnitude of the responses. According to these parameters, a greater response was observed during proestrus, compared with the other cycle stages. The significance of these results id discussed.


Erythroblastosis models. II. Materno-fetal incompatibility in chimpanzee. Erythroblastosis fetalis represents a significant hazard for successful management of pregnancy in man and in marmosets, but not in crab-eating macaques. Materno-fetal blood group incompatibility in chimpanzee is described as a contributing factor in the death of an infant. The findings indicate that parental blood groups should be taken into consideration when breeding chimpanzees.


[Endoscopic laser therapy of bladder tumors in man]. 15 patients with papillary bladder tumors were radiated with a Neodym-YAG laser beam since summer 1976. The seize of the tumors ranged from 5 to 10 mm in diameter. Neither complications nor tumor recurrency in the former tumor area could be observed. A final statement about the value of laser radiation in malignant tumors is not yet possible because of the small number of patients and the short control period. However, our preliminary experiences might be a beginning of a successful improvement of bladder tumor therapy by use of endoscopic laser radiation.


[Current status of gestagen administration. 2. Gestagen therapy in the area of reproduction]. The spectrum of progestin therapy has changed and expanded during the last few years. 1. The drug-therapy of choice in endometriosis is the medication of progestins for at least six months, for instance ethinyl-testosterone. If a patient wants additional children the "more gentle" dydrogesterone should be considered. 2. In the treatment of dysmenorrhea combination pills should be given, sequentials should be avoided. In the case of incompatibility of estrogens or in danger of oversuppression syndrome dydrogesterone should be applicated. 3. Dysfunctional bleedings should lead to an intense search for their cause. The treatment consists in an estrogen-progestin combination for 9 days and in cyclic continuation of this therapy for at least a further three months. In the case of hemorrhagic diathesis progestin treatment should be continued. 4. Cyclic adequate progestins have proofed to be successful in handling of hirsutism. The choice of the preparation depends on the patient's wish for children. 5. The progestin test is still the first step in diagnosis of amenorrhea. 6. Progestin therapy is indicated in progressive endometrial carcinoma. Some medical centres treat carcinoma of the mamma successfully with progestins. 7. Nowadays fast and early hormonal pregnancy tests are available. The progestin-pregnancy-test is limited to cases of premenopause. 8. The so-called short luteum phase has received considerable attention as a possible cause of infertility. In these cases a substitutional therapy of progestins should follow. Clomiphene or HCG-therapy is advisable. In short luteum phase and premenstrual spottings potent progestins should be given. 9. High dosage of progestins are in common use in the treatment of abortus imminens. 10. Combination pills and sequentials are widely used, the possible methods of a pure progestin contraception are: minipills, three-month-injections, implanted silastic capsules with progestional compounds, progestin impregnated intrauterine devices, vaginal silastic rings impregnated with progestional compounds. 11. Carcinogenesis of progestins was not detectable. 12. Some progestins are teratogenic.


Development of hemangiosarcomas in B6C3F1 mice fed 2-methyl-1-nitroanthraquinone. Subcutaneous hemangiosarcomas developed in 97% of 172 B6C3F1 mice of both sexes fed either 0.03% or 0.36% 2-methyl-1-nitroanthraquinone in the diet. There was no significant relationship to dose or sex. In addition similar vascular tumors occurred in the mesentery of 14 mice. 2-Methyl-1-nitroanthraquinone is carcinogenic in B6C3F1 mice when given in food. One of 97 control mice had a splenic hemangiosarcoma.


Comparative pressure-length-diameter relationships of neonatal and adult rabbit ureters. Pressure-length-diameter relationships of neonatal and adult rabbit ureters were studied in vitro. The increase in diameter, in response to an intraluminal pressure load of 10, 20, and 40 g per cm2, was faster and greater in the neonate than in the adult. Incubation in norepinephrine decreased the diametral deformation of neonatal ureters but had little effect on deformation in the adult ureter. The in vitro neonatal rabbit ureter, thus, appears to be more compliant and more sensitive to norepinephrine than the adult ureter.


The action of neuropharmacologic agents on the bladder and urethra during experimental spinal shock. Pressure recordings within the bladder, proximal (U1) and middle (U2) thirds of the urethra have been monitored in anesthetized female mongrel dogs before and just after being made paraplegic, i.e., during the period of spinal shock. In the intact animal, parasympathetic stimulation produces uninhibited waves whereas U2 pressure remains unchanged; during spinal shock, it produces a small rise of intravesical pressure, without uninhibited waves but with a marked increase in U2 pressure. This may explain why bethanechol chloride is not effective in producing bladder emptying during spinal shock. Stimulation of the beta-receptors of the synpathetic nervous system and blockade of the alpha-receptors both singly and in combination produce urethral "sphincter" relaxation during spinal shock. This might allow bladder evacuation during spinal shock with the Credé maneuver or abdominal straining.


Cobalt regulation of heme synthesis and degradation in avian embryo liver cell culture. Inorganic cobalt was found to induce heme oxygenase activity in primary cultures of embryonic chick liver cells and to inhibit the induction of delta-aminolevulinate synthetase by the porphyrinogenic compounds allylisopropylacetamide, dicarbethoxy-1,4-dihydrocollidine, etiocholanolone, phenobarbital, Aroclor (R)1254, and secobarbital. Much smaller concentrations of Co2+ (5 muM) were required to inhibit delta-aminolevulinate synthetase than to induce heme oxygenase activity (50 muM). These effects of Co2+ on heme synthesis and heme degradation were potentiated by depletion of cellular glutathione content as a result of treatment with diethyl maleate. Cobalt inhibition of the induction of delta-aminolevulinate synthetase was of the same magnitude and probably involved the same mechanism as that produced by cobalt heme dimethyl ester and iron heme. The induction of heme oxygenase by cobalt could be blocked by cycloheximide. Plasma protein synthesis was not inhibited in the presence of concentrations of Co2+ which produced inhibition of delta-aminolevulinate synthetase or induction of heme oxygenase. Other metals such as Cd2+ and Cu2+ also inhibited the induction of delta-aminolevulinate synthetase by allylisopropylacetamide. These findings indicate that Co2+ can regulate heme metabolism directly in liver cells without intermediate actions on extrahepatic tissues. It is suggested that regulation of production of delta-aminolevulinate synthetase and heme oxygenase is mediated through the action of the metal ion rather than the metal in the form of a tetrapyrrole chelate.


Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA. The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.


Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells. Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.


Isolation and characterization of transfer RNAs from Dictyostelium discoideum during growth and development. Transfer RNAs and aminoacyl-tRNA synthetases were isolated from vegetative and developing cells of the cellular slime mode Dictyostelium discoideum. Using the homologous synthetases, the tRANs were compared for their levels of acceptance of 17 amino acids. These levels were found to be the same. The tRANs were further compared by double label chromatography on reversed phase columns. Some major quantitative differences in individual peaks were observed. In addition, for a number of aminoacyl-tRNAs, small but reproducible displacements of individual peaks were apparent. In many cases the displacement was in the major peak, and in all but one case the developmental peak eluted ahead of the vegetative peak. Taken together, these results suggest that there is no change from growth to development in the transcription of tRNA genes, but that there are important changes in post-transcriptional modification.


Movement generated by interactions between the dense material at the ends  of microtubles and non-actin-containing microfilaments in Sticholonche zanclea. Axopods of the planktonic protozoan, Sticholonche, are used as oars to propel the organism through seawater. Within each axopod is an orgainzed array of microtubules which inserts into a dense material that assumes the form of the head of a hip joint. This material, in turn, articulates on the surface of the nucleus. Microfilaments, 20-30 A in diameter, connect the dense material associated with the microtubules to the surface of the nucleus, and they move the axopod by their contractions. The active phase of the movement may take as little as about 0.04 s and the recovery phase may take between 0.2 and 0.4 s. The microfilaments are not actin, as based on: (a) their small diameter, (b) the lack of decoration with heavy meromyosin, and (c) their ability to coil, spiral or fold during contraction. By the use of Thorotrast, we were able to demonstrate that the cell surface is deeply infolded, extending all the way to the hip joint. Here, there is a specialized membrane system that resembles the diad in skeletal muscle. From cytochemical tests and the use of ionophores and chelators, there is some evidence that the motile process may be controlled by calcium. This study demonstrates that, in at least one system, microtubules can be moves by contractile microfilaments attached to the dense material at there tips.


The role of the centriolar region in animal cell mitosis. A laser microbeam study. An argon ion laser microbeam (488 and 514 nm) was used to selectively irradiate one of the two centriolar regions of rat kangaroo Potorous tridactylis (PtK2) prophase cells in vitro. The cells were sensitized to the laser radiation by treatment with acridine orange (0.1-0.2 mug/ml). Ultrastructural examination of the irradiated centriolar regions demonstrated that the primary site of damage was the pericentriolar material. This result suggests that nucleic acid is present in the pericentriolar material. Behavioral and ultrastructural analysis demonstrated that cells with one damaged pericentriolar zone could undergo (a) nuclear membrane breakdown, (b) chromosome condensation, (c) metaphase plate formation, and (d) cytokinesis. However, the chromosomes neither separated nor exhibited any anaphase movements. Detailed ultrastructural analysis revealed the presence of kinetochore microtubules on both sides of chromosome mass and a lack of microtubules in the cytokinesis constriction. These results indicate that the pericentriolar material is important in spindle orgainization and essential for the formation of the interpolar microtubules.


The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro. Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.


Nondestructive distinction between aflatoxin B1 and ethoxyquin in thin-layer chromatography. A rapid and simple method has been developed for the nondestructive distinction between aflatoxin B1 and the feed antioxidant, ethoxyquin. These two chemicals exhibit similar RF values in certain solvent systems and produce a similar bluish fluorescence under long UV (366 nm) radiation. The method involves the in situ generation of fluorescence spectra of the respective thin-layer chromatography spots. Since it is nondestructive, the method affords ancillary study of the separated aflatoxins.


Twenty-four hour integrated concentrations of progesterone, 17-hydroxyprogesterone and cortisol in normal male subjects. The 24 h integrated concentrations of progesterone, 17-hydroxyprogesterone (17-OHP) and cortisol were determined in 5 male subjects ranging in age from 25 to 36 years. Using a nonthrombogenic catheter and a constant withdrawal pump, blood was collected for a period of 24 h in 30 min aliquots. All five subjects had similar 24 h integrated concentrations of cortisol (9.2 +/- 5.4). One subject had elevated 24 h integrated concentrations of progesterone (55.2 +/- 18.7) and 17-OHP (352 +/- 122) when compared to the mean integrated concentrations (progesterone: 24.9 +/- 4.7; 17-OHP: 109 +/- 33) of the 4 other subjects. The 30 min integrated concentrations showed a diurnal variation for both 17-OHP and cortisol but not for progesterone. The best correlation between cortisol and 17-OHP occurred when the concentration of 17-OHP 90 or 120 min earlier. No significant correlation occurred between cortisol and progesterone.


MMPI profiles of brain-damaged and schizophrenic subjects. MMPI mean profiles for matched groups of 50 brain-damaged and 50 schizophrenic Ss from a short-term hospital were compared. The profiles are quite different. F tests demonstrated that the Sc scale best differentiated the two groups at far beyond the .01 level, and scales F, PA and PT also differentiated the two groups beyond the .01 level. A cut-off point of 80 on the SC scale correctly classified 78% of the two groups. Neither the 2-9 nor the 1-3-9 profile occurred a sufficient number of times in either group to be of any help in diagnosis.


Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction. A simple technique for the condensation of cellular 5-hydroxytryptamine (5-HT) with formaldehyde gas is described. The technique, which is especially suited for quantitative cytofluormetric studies, involves the generation of formaldehyde gas from dry paraformaldehyde in a closed reaction vessel with the addition of a measured quantity of water. The fluorescence yield of 5-HT was tested at various humidities. Optimal results were obtained with the addition of 100 mg water to a 1000 ml reaction vessel containing 6 g of dry paraformaldehyde. A major advantage of the method if the fact that the humidity during the reaction can be precisely controlled. The fluorescence yield of 5-HT, tested over a 50 day period showed excellent reproducibility. The stoichiometry of the reaction was tested by comparison of cytofluormetic data with that obtained by analysing the 5-HT content of pooled mast cells with an independent biochemical method. A highly satisfactory correlation (r = 0.96) was obtained within the range of 0.1 to 4 pg of 5-HT per cell. The limit of sensitivity of the cytofluorometric method was found to be of the order of 10(-13) g, and was determined by the fluorescence blank of the mast cells. This contributes to between 10 and 30 per cent of the total fluorescence emission from mast cells containing about 0.2 pg of 5-HT.


Hormonal effects on canine prostatic explants in organ culture. Explants of canine prostate were cultured in a defined medium for periods of up to 5 days with and without added steroids. Testosterone and 5 alpha-dihydrotestosterone failed to maintain their histological integrity and induced a greatly increased formation of stromal elements. Epitestosterone and 5 alpha-dihydroepitestosterone were partially successful in maintaining epithelial height although secretory activity was not preserved. The only steroid that sustained epithelial height and secretory activity whilst keeping stromal growth at a minimum was 5 alpha-androstane-3 alpha, 17 alpha-diol. The three other epimeric androstanediols were ineffective.


The effects of two response-elimination procedures on reinforced and induced aggression. Pecks against a stuffed pigeon were reinforced according to a fixed-interval schedule for one group of pigeons and a variable-interval schedule for a second group. Red and green stimulus lights were alternately illuminated. Subsequently, food deliveries no longer occurred during one color (extinction). In the presence of the other color, food was presented only when no attack occurred for 30 sec. When attack produced food, all pigeons generally exhibited characteristic fixed-interval or variable-interval response patterns. Two birds in each group frequently exhibited postreinforcement schedule-induced aggression. Attack was reduced to low levels at approximately the same rate by extinction and differential reinforcement of other behavior. For birds that had previously exhibited schedule-induced aggression the initial reduction of attack during the second experimental phase was followed by induced attack immediately after food delivery in the differential-reinforcement-of-other-behavior component and upon onset of the extinction component, Either extinction or differential reinforcement of other behavior may eliminate reinforced aggression but may be relatively ineffective for reducing induced attack.


Structure of mitochondria and vacuoles of Candida utilis and Schizosaccharomyces pombe studied by electron microscopy of serial thin sections and model building. The structure of mitochondria and of vacuoles in Candida utilis and Schizosaccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell (Davison & Garland, 1975). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.


Locus of blue and near ultraviolet reversible photoreaction in the stages of conidial development in Botrytis cinerea. The effect of the blue and near ultraviolet reversible photoreaction on conidial development in Botrytis cinerea was studied by observing microscopically selected conidiophores. Conidiophore development was divided into six stages; when the developing conidiophores from stage 2 (i.e. a mature conidiophore) to stage 5 (i.e. a conidiophore with conidium initial) were exposed to blue light for a short time, conidiation was suppressed; the conidiophores already formed de-differentiated to 'sterile' conidiophores with sharply pointed tips.  The suppression of conidial development by blue light could be reversed by subsequent exposure to near ultraviolet light, and conidia then developed normally. This mycochrome system functioned reciprocally within the range of identified conidiophore developmental stages and near ultraviolet light acted only at the same development stage as was inhibited by blue light.


Intracellular distribution of virus-specific RNA in chick embryo cells infected with Japanese encephalitis virus. Japanese encephalitis virus (JEV) infected chick embryo (CE) cells were treated with 4 mug actinomycin D/ml and 5 mM-D-glucosamine at 2 or 3 h before harvesting. Production of JEV was not affected by the short-time treatment of these drugs. The radioactivity in virus-specific RNA in the glucosamine-treated cells was a-parently higher than in non-treated cells. Nuclear and cytoplasmic extracts were prepared from the JEV-infected cells pulse-labelled with 3H-uridine at 15 h after infection. Analysis of virus RNA in nuclear extracts on sucrose density gradients showed that most of the radioactivity was in 23S RNA, 26S RNA and 8 to 12S RNA. The radioactivity of virus RNA in cytoplasmic extracts was found in 42S RNA and RNA fragments sedimenting at less than 8S.


Effect of dietary raw soybean and soybean trypsin inhibitor on trypsin and chymotrypsin activities in the pancreas and in small intestinal juice of growing swine. Sixty-eight growing gilts with a 12 kg average initial weight were used in seven trials to study the effect of dietary raw soybean (Harosov) and SBTI (Kunitz soybean trypsin inhibitor) on pancreatic and small intestinal trypsin and chymotrypsin activities. A solvent-extracted, heated soybean meal (SBM) was used, cause reduced growth. Both a single-meal and continuous feeding of the raw soybean diet caused a decrease in the pancreatic trypsin and chymotrypsin activities. In contrast, to the rat and the chick, the pancreas of the pigs did not enlarge subsequent to consumption of the raw soybean or SBTI diets. Raw soybean feeding also resulted in an inhibition of the intestinal trypsin and chymotrypsin activities. This inhibiting effect was greater than that of the SBTI, especially the chymotrypsin-inhibiting effect. This suggested that soybean constituents other than the SBTI, such as the Bowman-Birk inhibitor, caused inhibition. In the pig the inhibition of the intestinal proteolysis may be a major cause of reduced growth when raw soybean is fed.


Quantitative requirements of the hatchling green sea turtle for lysine, tryptophan, and methionine. The quantitative requirement for the amino acids lysine, tryptophan, and methionine was determined for the hatchling green sea turtle (Chelonia mydas). Hatchling green sea turtles were fed synthetic diets of purified substances with the composition of the diet varying in the amount of lysine, tryptophan or methionine present. The lysine requirement was found to be 4.8% of the crude protein (N X 6.25) or 1.7% of the dry diet. The tryptophan requirement was found to be 0.63% of the crude protein or 0.22% of the dry diet. The methionine requirement, in the presence of adequate cystine (3.1% of the crude protein), was found to be 1.5% of the crude protein or 0.49% of the dry diet.


Diurnal changes in plasma and liver lipids and lipoprotein lipase activity in heart and adipose tissue in rats fed a high and low fat diet. In order to evaluate a) the respective roles of adipose and muscle lipoprotein lipase (LPL) in the clearing of alimentary lipemia and b) the role of the resulting nonesterified fatty acids (NEFA) in controlling hepatic ketogenesis and liver triglyceride content, a number of parameters related to lipid metabolism were studied over the 24 hour period (the dark period being from 1930 to 0730 hours), in rats ad libitum fed either a low-fat (LF) or a high-fat (HF) diet containing respectively 1.1% and 41.5% lard. During spontaneous feeding (from 1500-1800 hours onwards), LPL activity in LF rats increased in adipose tissue and decreased in heart; in rats fed the HF diet for 3 weeks, the postprandial rise in adipose LPL was smaller and there was no decrease in heart LPL before 2100 hours. In HF rats, unlike the LF, feeding resulted in a large increase in circulating NEFA and total ketone concentrations, an increase in liver beta-hydroxybutyrate dehydrogenase activity, and a decrease in hepatic triglyceride content. The findings clearly indicate that in HF rats, muscle LPL controls the postprandial rise in plasma NEFA concentrations, which in turn appear to determine the extent of ketonemia and liver triglyceride changes. The possible control of these metabolic events by insulin is discussed.


Utilization of analogues of riboflavin by the riboflavin-deficient chick embryo. Detailed studies of the biological activity of several analogues of riboflavin in the riboflavin-deficient chick embryo were undertaken to extend our knowledge of the comparative biochemistry of these flavins. The riboflavin-deficient eggs were produced by hens homozygous for an autosomal recessive gene (rdrd). 7-Ethyl-8-methyl-flavin was found to be an adequate replacement for riboflavin in che chick embryo, a finding which mimics its effect in the riboflavin-deficient rat. 7,8-Diethyl-flavin was found to be an antagonist in the chick embryo as had been found to be true in the rat. 7-Methyl-8-ethyl-flavin can not be used as a replacement for riboflavin in the chick embryo and further, it inhibits the utilization of riboflavin in normal eggs. 7-Methyl-8-ethyl-flavin had been found to be an adequate replacement for riboflavin in the riboflavin-deficient rat. The classes mammalia and aves are clearly different in respect to the utilization of this flavin.


Effects of fluoride on livestock.

Effects of fluoride on livestock. Animals normally ingest small amounts of fluorides in their diet with no adverse effect. An increased ingestion of fluoride can be harmful to animals, and grazing animals can be damaged by the consumption of high-fluoride vegetation. Cattle have been the species most commonly affected, and the symptoms of excessive fluoride ingestion in that species include; lesions in the developing dentition, skeletal lesions, lameness, chemical evidence of tissue fluoride ingestion in that species include: lesions in the developing take and decrease in production. The level of fluoride which can be tolerated by various species with no adverse effect has been determined, and this data can be used to set a standard to protect animals from the deleterious effect of fluoride ingestion.


Baeocystin in Psilocybe semilanceata.

Baeocystin in Psilocybe semilanceata. Baeocystin and psilocybin were found in extracts of a variety of Psilocybe semilanceata. Psilocybin (but not baeocystin) was also detected in extracts of a related species. Psilocyte pelliculosa. Traces of psilocin were present in these two species. The structures of the isolated compounds were corroborated using mass spectrometry and UV spectroscopy.


Effect of lysine and diethanolamine-rutin on blood levels, withdrawal reaction, and acute toxicity of ethanol in mice. The effects of L-Lysine and diethanolamine-rutin on blood ethanol levels and withdrawal convulsions following a 4-day exposure to ethanol vapors were evaluated in 30-40-g male Swiss-Webster mice. The animals were exposed in groups, along with simultaneous controls, in a 34-liter dynamic Plexiglas exposure chamber. Blood was collected by retro-orbital puncture and analyzed enzymatically for ethanol. At the end of the inhalation period, convulsions on handling were scored over 24 hr. In addition, the effects of L-Lysine and diethanolamine-rutin on blood ethanol levels and on acute ethanol toxicity following oral and intraperitoneal administration were evaluated. L-Lysine lowered blood ethanol levels following inhalation or oral administration of ethanol. Diethanolamine-rutin had no effect on blood ethanol levels. Both L-Lysine and diethanolamine-rutin decreased the withdrawal reaction in dependent mice. L-Lysine increased the oral LD50 of ethanol, while diethanolamine-rutin decreased the intraperitoneal LD50 of ethanol.


Use of rabbits for GI drug absorption studies. A novel procedure to control the stomach emptying rate in rabbits is presented. Rabbits were given a special solid diet for 1 week, and then the gastric contents were washed out with saline. Then the rabbits were muzzled to prevent coprophagy during the night. Fifty grams of special soft diet given to the "stomach-emptying-controlled" rabbit transferred exponentially from the stomach into the small intestine and almost disappeared from the stomach within 5 hr. Griseofulvin, indomethacin, or nalidixic acid was administered in a hard gelatin capsule or tablet, with subsequent feeding of a special soft diet. Good correlations were observed between the plasma level-time curves of these drugs in the stomach-emptying-controlled rabbits and in human subjects.


Effects of colchicine on the ovine corpus luteum: role of microtubules in the secretion of progesterone. By 4 h after i.v. injection of ewes at the mid-luteal phase of the oestrous cycle with colchicine (1 mg/kg), the concentration of progesterone in peripheral plasma was halved while the content of progesterone in luteal tissue was doubled. The ultrastructure of the luteal tissue showed some specific drug-induced changes: microtubules were no longer present and the intracellular transport and secretion of granules associated with progesterone secretion appeared to be inhibited.


The effect of oxytocin treatment on the levels of prostaglandin F in the blood of heifers. Six heifers with normal oestrous cycles were treated i.m. with 100 i.u. oxytocin on 3 consecutive days, commencing on Days 1-6 after oestrus, and the levels of prostaglandin (PG) F in posterior vena cava plasma were compared with pretreatment values. An increase of PGF in response to oxytocin was significantly influenced by day, with the greatest response occurring on Day 3 after oestrus. In an ovariectomized heifer the levels of PGF in posterior vena cava plasma increased 24 h after priming with oestradiol, but no further increase occurred after oxytocin injection. Peak levels of PGF were higher in the plasma of the posterior vena cava than in the jugular vein. Various storage conditions of the blood before centrifugation and freezing (--20 degrees C) produced significant differences in plasma levels of endogenous PGF, but storage experiments with added labelled PGF-2alpha indicated that the PG was stable in plasma and whole blood.


Antagonists of vitamin B6. Simultaneous and stepwise modification of the 2 and 4 positions. Methods for the simultaneous and stepwise modification of the 2 and 4 positions of vitamin B6 have been devloped and have been applied to the synthesis of several analogues of this vitamin. 3,alpha5-O-Dibenzylpyridoxol was converted to its N-oxide and was rearranged to an alpha2-hydroxy derivative with (CF3CO)2O. The 2,4-bis(hydroxymethyl) intermediate was oxidized (MnO2) to the 2,4-dialdehyde, which was converted by a Wittig reaction with triphenylmethylphosphorane to the 2,4-divinyl derivative. Removal of the benzyl groups with acid gave the 2,4-divinylpyridioxol analogue, which was phosphorylated in the 5' position to give the cofactor analogue. The 2-CH3 in the known vitamin B6 antagonists, 4-deoxypyridoxol and 4-vinylpyridoxal, was similarly modified to CH2OH, CHO, and CH = CH2. Modifications of the 2 position in the vitamin B6 antagonists are expected to be associated with changes in selectivity for enzymes in various tissues without a concomitant loss of biological activity, because of the well-established bulk tolerance in this position. Active analogues are expected to undergo, in vivo 5-phosphorylation, which is probably a prerequisite for their antagonist activity. Some of the compounds (e.g., the 2,4-divinyl analogue) have substantial growth-inhibitory activity for cultured mouse mammary adenocarcinoma. In contrast to that of the parent compounds, this activity was only partially reversed by pyridoxal.


Analogues of 8-azainosine.

Analogues of 8-azainosine. A convenient synthesis of 8-azapurine ribonucleosides substituted at the 6 position with thio, alkylthio, alkoxy, amino, and alkylamino groups is described. The reaction of 6-(methylthio)-8-azapurine (1) with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride in the presence of Linde AW-500 molecular sieve gave a 2:1 mixture of 2 and 3, respectively. This mixture was rearranged by heating with molecular sieve in refluxing toluene to give a 6:1 mixture of 2 and 3. Treatment of 2 or 3 with the appropriate nucleophiles at room temperature gave 6-substituted 8-azapurine ribonucleosides (7-substituted 2- or 3-beta-D-ribofuranosyl-3H-1,2,3-triazolo[4,5-d]pyrimidines) 4-13. The thione 11 rearranges to N-beta-D-ribofuranosyl[1,2,3]thiadiazolo[5,4-d]pyrimidin-7-amine (14) in the solid state or in solution. All of these compounds were cytotoxic to H.Ep. No. 2 cells in culture except the parent base, 8-aza-6-(methylthio)purine (1) and the 8-isomers (3,12, and 13). Three of these compounds-8azaadenosine (4), 8-aza-6-(methylthio)purine ribonucleoside (5), and 8-aza-6-(methoxy)purine ribonucleoside (7)-showed borderline activity in the leukemia L1210 system. The thiadiazolopyrimidine (14) showed activity at three dose levels.


Inhibition of acetylcholinesterase by thiamine. A structure-function study. Eighteen compounds related to thiamine were assayed for their inhibitory potency against electric eel acetylcholinesterase at pH 7.00 and 8.25 by Ellman's method. Data in the form of progress curves were fitted to the integrated form of the rate equation for linear, mixed-type Michaelis-Menten kinetics. The values of Ki thus obtained were compared in order to define the loci of inhibition on the thiamine molecule. It was found that the positively charged quaternary nitrogen atom is the primary locus of inhibition, while alkyl groups in the molecule play a secondary role through hydrophobic association with the enzyme. Protonation of the inhibitors was seen to be an important factor.


Cannabinoids. 2. Synthesis and central nervous system activities of some B-ring homocannabinoid derivatives and related lactones. The syntheses of some novel B-ring homocannabinoid derivatives 1,2, and 4 and related lactones 3 and 5 are described. Compounds 1-5 are 6,7,8,9,10,11-hexahydrodibenz[b,d]oxepins. CNS structure-activity correlations were determined using three rodent models. The potency and activity profile of these seven-membered ring compounds were compared with the corresponding six-membered ring homologues 6 and 7. A possible seperation of CNS activities was noted with compound 1. Unexpected pharmacological activity was discovered with lactone 3, which was about equipotent to 1-delta9-tetrahydrocannabinol. It was found that dimethylation at the 7 postition decreased CNS activity when the 6 position was either a carbonyl or methylene group.


Aporphines, 21. (1,2) Dopaminergic activity of aporphine and benzylisoquinoline derivatives. Synthesis of 8-hydroxyaporphines and 1-(hydroxybenzyl)-2-n-propyl-1,2,3,4-tetrahydroisoquinolines. The synthesis and physical properties of 8-hydroxyaporphine (3a) and 8-hydroxy-N-n-propylnoraporphine (3b) are described. The replacement of the rigid aporphine ring system by the more flexible benzyltetrahydroisoquinoline moiety, still containing all the necessary substitutents of the potent dopamine agonist N-n-propylnorapomorphine (1b) (NPA), resulted in the synthesis of 1-(3,4-dihydroxybenzyl)-2-propyl-1,2,3,4-tetrahydroisoquinoline (4). Analogous to 4,1-(4-hydroxybenzyl)-2-propyl-1,2,3,4-tetrahydroisoquinoline (5) was synthesized for a direct comparison with the biological activity of the corresponding 10-hydroxyaporphine (2). All compounds synthesized were evaluated as salts of their racemates. In animals with unilateral 6-OHDA lesion of the nigrostriatal pathway, (-)NPA and 2 caused dose-dependent contralateral circling behavior although activity was greatly reduced for the monohydroxylated aporphine 2. 3b,4, and 5 were inactive at doses of 0.25-4.0 mg/kg sc. Compounds 2 and 3b exhibited very weak activity in the stereotype tests in comparison to the response obtained with apomorphine and (-)-NPA. 4 and 5 failed to induce any sterotyped response. These compounds were also investigated for their ability to stimulate locomotor activity following direct injection into the nucleus accumbens. (-)-NPA induced a modest increase in activity but apomorphine completely failed to elicit a locomotor responses and antagonized the effect induced by dopamine. 2,3b, 4 and 5 neither enhanced locomotor activity on direct injection into the nucleus accumbens nor antagonized the hyperactivity response to intraacumbens dopamine when administered peripherally. On direct injection into the caudate-putamen only apomorphine induced stereotyped biting; (-)-NPA, 2, 3b, 4, and 5 were inactive. The differential activity of the aporphine derivatives in these tests strongly supports the possible existence of different types of dopamine receptors within the extrapyramidal and mesolimbic systems. The present studies confirm that the flexible benzylisoquinolines 4 and 5 do not adopt the active dopamine conformation and that the rigid aporphines, preferably containing hydroxyl functions at the 10 or 11 positions, are of greater importance in eliciting potent dopamine agonist activity.


Formation and structure of infectious DNA of spleen necrosis virus. The kinetics of formation and the structure of infectious DNA of spleen necrosis virus were determined. Nonintegrated infectious viral DNA first appeared 18 to 24 h after infection of dividing cells and persisted for more than 14 days. The nonintegrated infectious viral DNA was in the form of either a double-stranded linear DNA with a molecular weight of 6 X 10(6), detected in both the cytoplasm and nucleus, or a closed circular DNA of the same molecular weight, detected primarily in the nucleus. Integrated infectious viral DNA appeared soon after the nonintegrated infectious viral DNA and was the predominant form of infectious viral DNA late after infection. Integration of the spleen necrosis virus DNA into the chicken cell genome was demonstrated by three independent criteria. Nucleic acid hybridization indicated that the linear infectious viral DNA had a 5- to 10-fold higher specific infectivity than either the closed circular or integrated infectious viral DNA. Infectious viral DNA did not appear in infected stationary cells, indicating some cellular influence on the formation of infectious viral DNA.


Virus-specific protein synthesis in cells infected by infectious pancreatic necrosis virus. A study of virus-specific protein synthesis in infectious pancreatic necrosis virus-infected RTG-2 cells was undertaken to find a relationship between the coding capacity of virus genome (two segments of double-stranded RNA of 2.5 X10(6) and 2.3 X 10(6) molecular weight) and the sizes and relative amounts of polypeptides in the virion and in infected cells. The time course of virus-specific protein synthesis was followed by pulse labeling infected UV-irradiated cells with [35S]methionine and analyzing the labeled proteins by polyacrylamide gel electrophoresis followed by autoradiography. Three size classes of virus-specific polypeptides were synthesized, in the same relative proportion, throughout the infectious cycle, beginning 3 h postinfection. Their designation and molecular weight was as follows: alpha1, 1000,000; alpha2, 90,000; beta1, 59,000; beta2, 56,000; gamma1, 32,000; gamma2, 30,000; and gamma3, 28,000. Experiments using amino acid analogues, protease inhibitors, ZnCl2, and supraoptimal temperatures showed that polypeptides of the beta and gamma families did not arise from the alpha polypeptides by post-translational cleavage. Slow cleavage late in the infectious cycle could be demonstrated, since during 12-h period radioactivity was chased from beta1 via beta3 to beta4 (molecular weight 50,000) and beta5 (molecular weight, 49,000). During the chase most of gamma2 was degraded, whereas radioactivity could not be chased from the remaining virus-specific polypeptides. Purified virus contained polypeptides alpha1, alpha2, beta4, beta5, and gamma1. The beta polypeptides made up over 60% of the virion proteins. The results suggest that infectious pancreatic necrosis vibrus possesses a unique mechanism for synthesis of three size-classes of proteins using mRNA transcripts from two high-molecular-weight double-stranded RNA genome segments.


Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation. fu-1 cells, a nonfusing variant of the L8 line of rat myoblasts, form syncytia upon infection with murine leukemia virus (MuLV) or upon cocultivation with MuLV-infected cells; L8 cells do not form these syncytia, but do fuse into multinucleate myotubes. Syncytia of fu-1 cells form within 1 h after infection. The number of syncytia formed is proportional to the multiplicity of virus within a range of 4 to 16 and is maximum when the cell density is subconfluent. When either XC or fu-1 cells are productively infected with MuLV, they become resistant to syncytia formation by passage 3. The rapid formation of syncytia in fu-1 cells was found amenable for selection of temperature-sensitive mutants of MuLV and for screening additional variants of the L8 line.


Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon. We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.


Binding of adenovirus to microtubules. II. Depletion of high-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding. A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.