Formation of hybrid concanavalin A molecules by subunit exchange. Mixing of native concanavalin A (Con A) and its dimeric succinylated derivative (succinyl-Con A) in glycine-HCl buffer, pH 4.5, resulted in the formation of a new chemical species that could be separated as a unique fraction by DEAE-cellulose chromatography or by gel electrophoresis. The molecular weight of this new component, which contained the subunits (Mr= 26,000) of native Con A and its succinyl derivative in equimolar amounts, was 50,000 at both pH 5 and pH 7. These data suggest that subunit exchange between 2 chemically distinct Con A molecules yields a hybrid molecule consisting of one protomer of native Con A and one protomer of succinyl-Con A. Similar exchange reactions and hybrid molecules were also observed after mixing acetyl-Con A and succinyl-Con A. These procedures provide several new chemical variants of the Con A molecule that may be useful for the analysis of lectin-cell surface interactions.


Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Binding of radioiodinated peptide. We have used 125I-labeled vasoactive intestinal peptide (VIP) to study the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acinar cells prepared from guinea pig pancreas. Binding of 125I-VIP to pancreatic acinar cells was moderately rapid, reversible, specific, saturable, and depended on incubation temperature. Deterioration of 125I-VIP incubated with pancreatic acinar cells at 37 degrees was reflected in a decrease in acid-precipitable radioactivity and in the amount of tracer which could bind to fresh acinar cells. On the other hand, 125I-VIP bound to pancreatic acinar cells appeared to be protected from deterioration. VIP and secretin but not glucagon or COOH-terminal octapeptide of cholecystokinin inhibited binding of 125I-VIP to pancreatic acinar cells. The dose-response curve for inhibition of 125I-VIP binding by VIP or secretin was biphasic and suggested that pancreatic acinar cells have two classes of binding sites: (a) a relatively small number of sites with a high affinity for VIP and a low affinity for secretin, and (b) a relatively large number of sites with a low affinity for VIP and a high affinity for secretin. The difference between the relative affinities of VIP and secretin for the high affinity VIP binding sites appears to be primarily attributable to the NH2-terminal portions of these molecules since synthetic COOH-terminal fragments VIP 14-28, VIP 15-28, and secretin 14-27 were equipotent in inhibiting 125I-VIP binding. On the other hand, secretin 5-27, [6-tyrosine] secretin and native secretin were equipotent in inhibiting binding of 125I-VIP to its high affinity site, and these three peptides were 5 times more potent than secretin 14-27 but 10,000 times less potent than native VIP.


Separation, purification, and properties of multiple forms of cytochrome P-450 from the liver microsomes of phenobarbital-treated mice. Four distinct cytochrome  P-450 fractions (A1, A2, C1, and C2) have been separated and purified from the liver microsomes of phenobarbital-treated hybrid mice (B6D2F1/J). Fractions A2 and C2 were highly purified with specific contents of 16.5 and 17.5 nmol of cytochrome P-450/mg of protein, respectively, based on their amino acid compositions. The major hemeprotein bands of A2 and C2 have different minimum molecular weights (50,000 and 56,000, respectively) on polyacrylamide gels in the presence of sodium dodecyl sulfate. All four fractions with respect to their spectral and catalytic properties, thereby demonstrating that mouse liver microsomes from phenobarbital-treated hybrid mice contain at least four forms of cytochrome P-450.


The isolation of eukaryotic ribosomal proteins. The purification and characterization of the 40 S ribosomal subunit proteins S2, S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28. The proteins of the small subunit of rat liver ribosomes were separated into five groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5 (Collatz, E., Lin, A., Stöffler, G., Tsurugi, K., and Wool, I.G., (1976) J. Biol. Chem. 251, 1808-1816). From the several groups, 12 proteins (S2,S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28) wereisolated by ion exchange chromatography on carboxymethylcellulose, by chromatography on sulfopropyl-Sephadex, and by gel filtration through Sephadex G-75. The amount of protein obtained varied from 1 to 9 mg depending on the number of steps required for the preparation; several proteins had no detectable contamination and the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyazrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.


Characterization of mannose-labeled glycopeptides from human diploid cells and their growth-dependent alterations. Mannose-labeled glycopeptides were prepared from human diploid fibroblasts harvested by brief pronase digestion. Combined use of endo-beta-N-acetylglucosaminidase H and D converted most of the mannose-label into arrays of oligosaccharides. They were separated by paper chromatography and were characterized by Sephadex G-25 column chromatography, by affinity column chromatography on concanavalin A-Sepharose, and by successive digestion with alpha-mannosidase and beta-mannosidase. The results indicated that mannose residues existed as clusters of various sizes, which we refer to as "oligomannosyl cores". The large oligomannosyl cores (approximately 7 to 8 mannosyl residues) were predominant in the glycopeptides from growing cells and were preferentially associated with neutral glycopeptides, similar to Unit A glycopeptides of thyroglobulin (mannose-N-acetylglucosamine unit). In glycopeptides from nongrowing cells, the ratio of the large oligomannosyl cores decreased, accompanying the increase of a small oligommanosyl core consisting of 3 mannosyl residues. The small core was preferentially associated with acidic glycopeptides.


Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.


Effects of estrogen on gene expression in chick oviduct. The role of chromatin proteins in regulating transcription of the ovalbumin gene. Histones, extractable non-histone proteins, and a tightly bound non-histone protein DNA complex were fractionated from chromatins which were isolated from estrogen-stimulated and hormone-withdrawn chick oviducts. Reconstitution of homologous constituents was performed and RNA was transcribed from these reconstituted chromatins using Escherichia coli RNA polymerase. DNA complementary to ovalbumin mRNA was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences in these in vitro transcripts. Our results demonstrated that 0.011% of the in vitro RNA transcripts produced from reconstituted estrogen-stimulated chromatin was ovalbumin mRNA sequences as compared to 0.0015% obtained for reconstituted hormone-withdrawn chromatin. Thus, the ratio of ovalbumin mRNA sequences in the in vitro transcripts of reconstituted stimulated and withdrawn chromatins was 8 to 1, identical with the value obtained from native estrogen-stimulated and hormone-withdrawn chromatins. Furthermore, the number of initiation sites for RNA synthesis on reconstituted and native chromatins are indistinguishable. Thus, the specificity of transcription of isolated chromatins appears to be conserved upon dissociation and fractionation of the chromatin proteins followed by reconstitution of these constituents to DNA. The effect of chromatin proteins on gene expression was further examined by reconstituting components from different development stages. Reconstitution of extractable non-histone proteins from estrogen-stimulated chromatins to tightly bound non-histone protein. DNA complexes from hormone-withdrawn chromatins resulted in the synthesis of a substantial amount of ovalbumin mRNA sequences. Conversely, when extractable non-histone proteins from withdrawn chromatins were reconstituted to tightly bound non-histone protein DNA complexes from estrogen-stimulated chromatins, very low levels of mRNAov sequences were detected. In contrast, interchange of the histones and tightly bound non-histone protein DNA complexes from hormone-withdrawn and estrogen-stimulated chromatins during reconstitution did not affect the level of mRNAOV sequences produced. Therefore, the extractable non-histone proteins of chromatin appear to be extremely important in regulating specific gene expression.


Carboxamidomethyl esters as reactive substrates for alpha-chymotrypsin. Orientational effects of hydrogen-bonding interactions. Effects of hydrogen-bonding interactions of amide groups on reactivity of esters to alpha-chymotrypsin were studied. Of the methyl esters studied, only that from acetyl-L-phenylalanine has k3 rate-limiting. In methyl beta-phenylpropionates an alpha-acetamido substituent increased k2 greater than 550 times, k3 approximately 5 times; an alpha-acetylclycyloxy substituent increased k2 approximately 2 times, k3 approximately 6 times, both in comparison with the alpha-acetoxy esters. Essentially all carboxamidomethyl esters studied have k3 rate-limiting; reactivity to hydroxide is only 4 times that of methyl esters. In alpha-substituted beta-phenylpropionates, carboxamido-methyl esters show k2 values greater than 110 times greater than 280 times, greater than 26 times, and 7 times the k2 values of the methyl esters for the alpha substituents, acetoxy, acetylglycyloxy, hydroxy, and hydrogen, respectively. In esters of alpha-acetamido acids, carboxamidomethyl esters show k2 values 44 times, greater than 110 times, greater than 12 times, and approximately 33 times the k2 values of the methyl esters of glycine, alanine, leucine, and phenylalanine, respectively. Cyanomethyl esters also had k3 rate-limiting. Hydrogen-bonding to the enzyme of either an alpha-acetamido group or a carboxamidomethyl group combined with bonding of the beta-aryl group, orients the hydrolyzing groups properly, increasing k2. Hydrogen-bonding of both alpha-acetamido and carboxamido-methyl groups is effective to a lesser degree. The amide group appears to have small effects on Ks as hydrogen bonding is balanced by desolvation. It is proposed that desolvation during bonding increases k2 and Ks.


Organization of membrane proteins in the intact myelin sheath. Pyridoxal phosphate and salicylaldehyde as probes of myelin structure. Pyridoxal phosphate and salicylaldehyde were used as protein-labeling probes to study the organization of membrane proteins in the intact myelin sheath of the cat dorsal column. Both reagents react with protein amino groups to form Schiff's bases which can be reduced with NaB3H4. The relatively membrane-impermeant pyridoxal phosphate labels all proteins of the intact myelin except basic protein. This major protein of myelin is labeled only after loss of membrane integrity. The relatively membrane-permeant probe, salicylaldehyde, was then used to establish that the basic protein is truly located on the cytoplasmic side of the myelin bilayer, and not merely sequestered within the multiple lamellar structure of the sheath. All proteins in the intact myelin are readily labeled by this reagent, with the label distribution pattern identical to that of disrupted myelin fragments. These data suggest a model for myelin structure in which the basic protein is the only major protein component located exclusively on the cytoplasmic side of the membrane (the major period zone of the sheath), with the other major proteins disposed wholly, or in part, in the extracellular half of the membrane bilayer (the intraperiod zone). All proteins, although asymmetrically disposed with respect to membrane sidedness, appear to be randomly distributed throughout the lamellae which comprise the sheath.


Regulation of adenylate cyclase from glial tumor cells by calcium and a calcium-binding protein. A biphasic response to changes in Ca2+ concentration was observed for basal and norepinephrine-stimulated adenylate cyclase activity in homogenates of C-6 glioma cells. The enzyme was stimulated approximately 40% by low concentrations of free Ca2+ (less than or equal to 1 muM) and inhibited to successively greater extents as free Ca2+ concentrations were increased to approximately 100 muM. Ca2+ did not alter the concentration of norepinephrine required for enzyme activation. Homogenates of C-6 cells were separated into particulate and supernatant fractions by centrifugation at 27,000 X g for 20 min. The particulate fraction contained nearly all of the adenylate cyclase activity. This activity was stimulated approximately 40% by the addition of untreated supernatant fraction, by boiled or dialyzed supernatant fraction, and by a homogenous Ca2+-binding protein (calcium-dependent regulator (CDR) prepared from brain. Addition of either the supernatant fraction or CDR lowered the Ca2+ concentration required for maximal stimulation of the adenylate cyclase. The factor in the supernatant fraction which activated the particulate enzyme was subsequently identified in acrylamide gel electrophoretic studies to be CDR. The amount of CDR required for maximal activation of the enzyme was found to be lowered as the Ca2+ concentration in the assay was increased. High amounts of added CDR (100 to 1000 ng) were inhibitory. Use of the monionic detergent, Lubrol PX, to prepare dispersed adenylate cyclase from the particulate fraction resulted in large losses of activity. The resultant preparation of enzyme contained some CDR which could not be removed by chromatography of the preparation on anion exchange columns. Addition of homogeneous CDR to the assay activated the enzyme several-fold at low Ca2+ concentrations. At higher Ca2+ concentrations the enzyme was activated fully by the CDR endogenous to the preparation and added Ca2+. CDR was inhibitory.


Titration of the carboxyhemoglobin tetramer-dimer equilibrium by inositol hexaphosphate. The results of a series of light scattering experiments of the reaction of inositol hexaphosphate (at pH 7.0) over 6 orders of magnitude of concentration (10(-8) to 10(-2) M) with carboxyhemoglobin indicates that there is a shift in the tetramer-dimer equilibrium towards the tetramer, reaching a maximum effect at 0.1 mM inositol hexaphosphate. Raising the phosphate concentration beyond this latter value promotes dissociation to dimers. However, in this range some of the dissociation of carboxyhemoglobin was undoubtedly due to the increase in ionic strength from the inositol hexaphosphate ion. If the effect of ionic strength is allowed for by classical Debye-Hückel theory, one- and possibly two-phosphate binding sites per dimer can be detected. (Approximate association constant is 8000 M-1 for a single site at 0.1 ionic strength). The location of such sites is considered to lie near the dissociable plane of the hemoglobin tetramer and possibly to include half of the residues that bind inositol hexaphosphate in the tetramer.


Crystallization and preliminary crystal data of iron-containing superoxide dismutase from Pseudomonas ovalis. Large single crystals of iron-containing superoxide dismutase from Pseudomonas ovalis were prepared from 50% saturated ammonium sulfate solution, at pH 4.5, on gentle evaporation of the solvent at 4 degrees. The crystals were monoclinic, space group P2, with unit cell dimensions a = 81.9 A, b = 49.0 A, c = 61.0 A, and  beta = 106 degrees. Considerations of cell volume and protein molecular weight indicated 1 molecule of superoxide dismutase in the assymmetric unit, the smallest number reported so far for superoxide dismutases.


Bone ingrowth into dynamically loaded porous-coated intramedullary nails. Research to determine whether porous-coated Vitallium intramedullary rods could be used to bridge segmental defects in long bones was performed using rabbit tibias as a suitable model for human bone growth. One-centimeter segments of the midshaft of tibias of mature rabbits were removed surgically and replaced with equal-sized segments of Silastic tubing to maintain leg length. A coated rod was inserted through the proximal tibial segment, through the tubing, and into the distal bone segment. The legs were taken out of plaster at 3 weeks, and x-rays were taken periodically until sacrifice. At sacrifice, 30 weeks postoperatively, the mechanical properties of both tibias from each rabbit were measured destructively in a torsional testing machine. The load at failure of the tibia with the segmental defect averaged 90% of the contralateral control tibia. Rod pullout data and electron-probe x-ray micro-analysis indicated that a substantial rod-endosteal bone bond existed due to ingrowth into the porous coating. The torsional data may have been somewhat obscured by the presence of bony callus which grew over the Silastic tubing and bridged the segmental defect exosteally in every case.


Animal studies on bone ingrowth kinetics of ceramic material under dynamic stress. Two of the primary problems of implants in use today result from the materials used in their construction (metal and polyethylene) and from the necessary additional fixation with bone cement. In order to alleviate these well known difficulties, we studied ceramic material (99.7% Al2O3 with 0.25% MgO) which exhibits several advantages in these areas, but has a diminished bending strength. To take advantage of the ceramic material, we tested a cement-free implantation which should allow unhindered growth of bone tissue to or into the material. In particular, the interface relationship under load bearing was investigated. The course of the tissue differentiation and ingrowth on the surface of the ceramic implants under dynamic stress, was studied by a specifically disegned distance-spacer. These were tested on the femur of foxhounds and sheep, as well as by use of temporary immobilization. Histological investigations in intervals of 4 weeks showed the course of the development of the interface tissue. After removal of the binding materials, the behavior of the implant support is studied in 4-week intervals with free load bearing. Radiological and histological development studies are shown. The results we obtained implicate the use of ceremic impants. But the application in humans still seems to be problematic, as there is a need for designs which are loaded by pressure only.


Chemical and mechanical behavior of bioglass-coated alumina. This work has shown that it is possible to produce a bioglass-coated, high-strength, fully dense alumina implant system with a variable surface reactivity. Furthermore, it has been demonstrated that this system will bond with bone. These two factors, plus the inherent strength of the dense alumina, make this system an attractive candidate as a new prosthetic material.


Evaluation of a bioglass-coated Al2O3 total hip prosthesis in sheep. A bioglass-coated fully dense alumina total hip was evaluated in sheep without using polymer bone "cement." After 3 months following surgery, both the acetabular cup and femoral stem component were firmly attached to bone. Histologic and radiographic analysis of the bone-implant interface showed regions of woven and even lamellar bone formation where high load transmission occurred. In regions of low loading or poor fit, a pseudosynovial fibrous lining was present.


Biomechanically induced tissue reactions after Al2O3-ceramic hip joint replacement. Experimental and early clinical results. A biomechanical concept of interface maturation around cement-free implanted alumina-ceramic hip endoprostheses in sheep is explained. Provided there is rigid primary stability of the implant within the bone bed, hard tissue formation along the support areas of the implant is mainly dependent on implant shape and main  local stress direction. Three subsequent phases of interface maturation can be distinguished, finally resulting in firm fixation of the implant by mechanical interlocking of supporting bone and ceramic. A new composite alumina-metal hip endoprosthesis for partial cement-free implantation (cup) is presented. The technique, postoperative care, and encouraging early results in 5 patients are demonstrated. A new ceramic femoral component for cement free implantation is proposed.


Prefabricated silicone subdermal mandibular implants: observations and problems related to their application. Success in the use of custom-fabricated Silastic implants in patients with facial contour defects led us to utilize this method in reconstructing mandibular defects. However, mandibular reconstruction with alloplastic material is quite difficult because of the presence of continous stress and motion in that region. The evolution in the use in the use of the mandibular implant from the commercially available one to the K-wire reinforced, prefabricated, silicone implant was largely dependent on the availability of silicone and the ultimate result obtained. In the past few years, about 30 subdermal implants were inserted. Some of those implants were placed immediately into patients undergoing resection for tumor. The implant was prefabricated prior to surgery from x-ray measurements of the anticipated segment to be resected; it was then used to reconstruct the defect prior to the final closure of the operative wound. Other implants were manufactured 6-18 months after resection for tumor or for deformity as a result of trauma. The implants were used to reconstruct the facial defects and to correct the funtional abnormality, such as drooling or deviation of the affected side by the unstable mandibular segment. Patients with severe congenital malformation have soft tissue deficiency in association with their bony malformation. Therefore, implants in such conditions were doomed to failure. Patient selection is the key to successful implantation.


Postyield behavior of subchondral trabecular bone. Cylindrical specimens of bovine subchondral trabecular bone were tested to uniaxial compressive strain levels of 75% to study energy absorption during pore collapse. Stress-strain curves were characterized by macroscopic yield at about 8% strain followed by a significant horizontal pore collapse regime. Energy absorption occurred largely in this postyield regime. Yield strength and energy absorption capacity were found to increase linearly with specimen apparent density. Microstructural analysis of the deformed specimens verfied that the mechanism for energy absorption was primarily fracture and buckling of trabeculae. The results suggest that during fracture, the collapse of trabecular bone (and the consequent absorption of energy) serves to attenuate stresses transmitted through the skeleton and thus protect vital structures such as the brain.


Biocompatibility studies on surgical-grade titanium-, cobalt-, and iron-base alloys. An animal model system for the investigation of the influence of in vivo-produced metallic corrosion products upon tissues has been developed. Externally applied electrical stimulation produces the corrosion over a short period of time, and a poststimulation  period is allowed for tissue stabilization. Tissue reaction adjacent to the metallic alloys shows fibrous tissue capsules and metallic corrosion product contamination. These preliminary studies show that this animal model and methodology may provide a technique for investigating long-term tissue biomaterial interactions and the influence of iron (316L), cobalt (Vitallium), and titanium (Tivanium) alloy corrosion products upon the susceptibility of tissues to various biocompatibility challenges.


A simple in vitro screening test for blood compatibility of materials. An in vitro closed-cell kinetic blood-coagulation test was developed and used to evaluate the blood compatibility of materials. The test compares the clotformation rate on a test surface to that of a control material, Silastic. This method avoids anticoagulants, blood flow variations, and the exposure to air and other foreign surfaces. Eight pairs of cells are sequentially evaluated as to the weight of thrombus formed, the amount of unclotted blood, and the reduction in platelet count of the blood exposed to both the test materials and the control. These data are used to calculate a relative index whereby materials can be rated quantitatively as to the rate of clot formation on their surface. The order of decreasing clot formation on one group of materials studied was as follows: biolized (Imai et al., Trans. Amer. Soc. Artif. Int. Organs, 17, 6, 1973) poly (ether urethane), segmented polyurethane, formaldehyde-treated pericardium, Hydron, glutaraldehyde-treated pericardium, biolized natural rubber, Hexsyn, heparinized natural rubber, Silastic, and natural rubber. Urethane and aldehyde-treated natural tissue had a lower percentage of red thrombus formation. These results generally correlate with in vivo observations.


Design and biomechanical evaluation of a cementable endosteal blade implant. A cementable endosteal blade implant has been developed and evaluated. Inherent in the design are the following factors: minimization of stress concentrators, ease of implantation, and high resistance to loosening. Other potential advantages of this design as compared with conventional endosteal blade implants include reduced hazards of postoperative infection and reduced likelihood of metallic corrosion. Six conventional endosteal implants and six cementable implants were installed in steer mandibles using standard dental surgical techniques and Simplex-brand methyl methacrylate bone cement. They were loaded in uniaxial compresstion at a loading rate of 0.0122 in./min. the stiffness (S), deformation at 900-lb load (D900), proportional limil (PL), and load at 0.04 in. deformation (L0.04) were calculated for each test. The cementable design showed a twofold increase in stiffness, only 17% of the deformation at 900lb, more than twice the proportional limit, and at least twice the load at 0.04 in. deformation when compared with similar values for the conventional endosteal implants. This study reveals that, in addition to being unstable in bone, conventional endosteal implants are also unstable when used with acrylic bone cement. The new design should eliminate most of the problems associated with endosteal blade implantation.


Bone growth into porous high-density polyethylene. The purpose of this study was to delineate the process by which bone comes to fill the pores of porous high-density polyethylene (PHDPE) implants. PHDPE (450 mu pore size) pellets 4 mm in diameter and 1 cm long were implanted into the femurs of dogs. A bone biopsy procedure was utilized to obtain PHDPE pellets implanted for periods of 3 days through 8 weeks. A one-year biopsy specimen taken from the PHDPE coating on the stem of a canine total-hip prosthesis was also studied. The results demonstrated that significant amounts of bone formed within the PHDPE pellets as early as 14 days after implantation. Bone was identified throughout the specimens after 4 weeks. After 6 weeks, the tissue in hematopoietic marrow. Scanning electron microscopy was utilized  in conjunction with light microscopy and microradiography to study the ultrastructural features of the bone ingrowth process.


Structure and function of the human patella: the role of cancellous bone. Total joint and other prosthesis often require the removal and replacement of considerable quantities of cancellous bone, and often are anchored in place by grouting into cancellous bone. Thus, a possible source of failure or loosening of many types of prostheses may be rooted in the lack of understanding of the structure, properties, and function of this material. In addition, as we have pointed out before, cancellous bone may play an important biomechanical role in the etiology of joint degeneration. With these considerations in mind, the architecture of the cancellous bone in the human patella was studied by serial sectioning and microradiography, using an improved technique developed in our  laboratory. Volumes of cancellous bone with apparently different functional roles were identified. Stereological techniques were used to quantify the structural characteristics and geometrical relationships throughout the patella. These results led to a structural model for the cancellous bone of the patella, and a comprehensive picture of the internal architecture. The distribution of mechanical compliance and yield stress was also measured, as a function of location and orientation, by a specially constructed microcompression testing machine. The measurements reflected the distribution of trabecular architecture, and both the properties and architecture reflected the gross biomechanical function of the patella. Furthermore, a true structure-function relationship was derived. Patellar contact area studies were performed on fresh cadavers and mapped for various angles of flexion. The variation in contact areas and the spatial variations in stiffness are discussed relative to the biomechanics and clinical aspects of the patella.


Interaction effects among cortical bone, cancellous bone, and periodontal membrane of natural teeth and implants. Following the initial successes of Goldberg and Gershkoff (Dental Digest, 5, 11, 1947) with the placement of subperiosteal implants, interest in all types of implants emerged. As an aid to the evaluation of various designs and materials without resorting to clinical testing, finite element analyses are being conducted by a number of researchers to determine the stress system induced in bone. The present study investigates the effects of variation in the thicknesses of the periodontal membrane and cortical bone and of the model boundary on the stresses developed around a natural tooth or a tooth-shaped implant. The results show that strong effects due to the variation in these parameters can be expected. As a consequence, the problem of the analysis of any single implant must consider additional factors. These can influence overall implant design and may eventually help to explain what could appear as anomalies in clinical test results.


Poly(methyl methacrylate)--aqueous phase blends: in situ curing porous materials. A potential implant material has been developed which combines the advantages of curing in situ and an open porous structure. This paper deals with synthesis and characterization of the material, which is a modification of the well-known acrylic bone cements. Some early results of animal experiments are mentioned.


The fracture mechanics of fatigue crack propagation in compact bone. The purpose of this investigation was to apply the techniques of fracture mechanics to a study of fatigue crack propagation in compact bone.  Small cracks parallel to the long axis of the bone were initiated in standardized specimens of bovine bone. Crack growth was achieved by cyclically loading these specimens. The rate of crack growth was determined from measurements of crack length versus cycles of loading. The stress intensity factor at the tip of the crack was calculated from knowledge of the applied load, the crack length, and the specimen geometry. A strong correlation was found between the experimentally determined crack growth rate and the applied stress intensity. The relationship takes the form of a power law similar to that for other materials. Visual observation and scanning electron microscopy revealed that crack propagation occurred by initiation of subcritical cracks ahead of the main crack.


Effect of fluoxymesterone on the pituitary-gonadal axis: the role of testosterone-estradiol-binding globulin. Four normal men and two agonadal men were given the oral synthetic androgen, fluoxymesterone (9alpha-fluoro-11beta, 17beta-dihydroxy-17alpha-methyl-4-androstene-3-one) for three days. Plasma testosterone (T), 17 alpha-hydroxyprogesterone (17-OHP), and LH were measured every 30 minutes on a control day and on the first day of treatment. Testosterone and LH were measured every six hours on the last day of treatment. During the first 24 hours of treatment the number of LH secretory episodes per day decreased from 10.5+/-2.5 (SD) to 6.2+/-2.9 (SD) (P less than 0.01), mean 24 hour LH decreased from 12.6+/-3.5 (SD) mlU/ml to 9.3+/-3.7 (SD) mlU/ml (P less than 0.01), and mean 17-OHP decreased from 2.33+/-1.4 (SD) ng/ml to 1.18+/-0.39 (SD) ng/ml (P less than 0.01) in all normal subjects. T was significantly decreased (P less than 0.01) from 464.5+/-76.4 (SD) ng/100 ml to 294.2+/-99.5 (SD) ng/100 ml. By the third day of treatment, LH had decreased further in four, and T in three of four normals. The number of LH spikes and the mean LH levels did not decrease in the agonadal patients. In vitro, using equilibrium dialysis, fluoxymesterone displaced T from plasma binding proteins with an apparent K=1.0 x 10(8) and 1.9 x 10(7) in female and male plasma, respectively, at 22 C; and 5.2 x 10(7) and 8.0 x 10(6) at 37 C. In polyacrylamide gel electrophoresis, a 500-fold molar excess of fluoxymesterone decreased the peak of TeBG-bound T by 45% (P less than 0.01). The in vitro data are consistent with the possibility that, in vivo, the displacement of T from TeBG by fluoxymesterone may play a role in the suppression of the pituitary-gonadal axis by synthetic oral androgens in vivo.


Changes in serum triiodothyronine, thyroxine, and thyrotropin during treatment with thyroxine in severe primary hypothyroidism. Studies were performed on 40 patients with severe primary hypothyroidism, during treatment with varying doses of T4. Therapy was initiated with 50 mug/day and was continued for at least 2 months. Subsequent repeated increases of 25 mug/day were continued for at least 2 months until the serum TSH level was less than 10 muU/ml. Measurements of serum T4 and T3, RT3U, and serum TSH were carried out at monthly intervals. TRH tests were performed after TSH levels of less than 10 muU/ml had been attained and the dose of T4 had been maintained for at least 2 months. In most of the samples obtained during treatment with various doses of T4 (50-175 mug/day), serum T4 concentrations were within the normal range, even when patients were receiving only 50 mug/day of T4; however, approximately 60% of the samples had subnormal T3 concentrations. Fifty per cent of the samples had elevated TSH concentrations despite normal T4 levels, while only 7% of the samples with normal T3, as well as T4, levels had slightly elevated TSH concentrations. Similar relationships were observed between serum TSH and free T4 indices and free T3 indices. Among patients with serum TSH levels of less than 10 muU/ml, none showed subnormal T4 concentrations, while subnormal T3 concentrations were found in some of them. Hyper-response to TRH was noted in patients with subnormal T3 levels, and normal responses were observed in patients with normal T3 and T4 concentrations. These data indicate that, during the treatment of hypothyroidism with T4, 1) normal basal TSH correlates better with normal serum T4 and T3 than with normal T4 alone, 2) the response of TSH to TRH is supranormal in patients with subnormal T3 levels, in spite of normal basal TSH and T4, and 3) the calculated maintenance dose of T4, which is associated with a normal TRH response, is 2.08 mug/day/kg of body weight.


Circadian rhythm and effect of posture on plasma aldosterone concentration in primary aldosteronism. The effect of circadian rhythm and alterations in posture on plasma aldosterone concentration was studied in 13 patients with primary aldosteronism (six adenoma, five idiopathic hyperplasia, two carcinoma) to define the regulatory mechanism in each of these pathologic subtypes. Blood samples for aldosterone, cortisol, renin, and potassium concentrations were obtained every 4 h during prolonged recumbency (32 h) and upright posture (16 h). During recumbency, aldosterone and cortisol followed a normal circadian pattern in patients with adenoma and hyperplasia, with peak values at 0400-0800 h and the nadir at 1600-2400 h. Normalized aldosterone and cortisol values correlated significantly in both groups (adenoma r=+0.66, P less than 0.001; hyperplasia r=+0.42, P less than 0.01). With upright posture, aldosterone levels declined parallel to the normal circadian fall in cortisol in patients with adenoma (r=+0.68, P less than 0.001); whereas aldosterone levels increased in patients with hyperplasia parallel to small increments in renin (r=+0.65, P less than 0.001) and potassium (r=+0.64, P less than 0.001). During the administration of dexamethasone, aldosterone no longer correlated with cortisol in patients with adenoma but continued to correlate with renin during upright studies in patients with hyperplasia (r=+0.77, P less than 0.01). Aldosterone circadian rhythm was abnormal in patients with carcinoma and no effect of posture was noted. Unilateral adrenalectomy restored the normal postural relationship in four patients with adenoma. These studies suggest that aldosterone secretion is under continuous ACTH control regardless of posture in patients with adenoma, whereas persistent adrenal responsiveness to small increments in renin and/or potassium mediate the postural increase in plasma aldosterone in patients with hyperplasia. True adrenal autonomy occurs only in patients with adrenal carcinoma and when ACTH is suppressed in those with adenoma.


Pituitary function in pseudocyesis.

Pituitary function in pseudocyesis. Neuroendocrine assessments were made in a 16-year-old girl with pseudocyesis of 38 weeks duration. Basal levels of pituitary LH and PRL, but not of FSH, were markedly elevated and the pulsatile pattern of LH and PRL appears to be exaggerated compared with those found in normal cycling women. Growth hormone levels were normal, with an appropriate increase after arginine infusion and l-dopa. The increased luteotropic action resulting from increased plasma LH and the lactogenic action of increased plasma PRL were associated with the presence of luteal function and galactorrhea. The dominant role of psychic mechanism(s) in causing hypersecretions of LH and PRL is suggested by the almost immediate fall in the serum concentrations and the rapid resolution of abdominal distention after the diagnosis was revealed to the patient.


Effect of antithyroid agents 6-propyl-2-thiouracil and 1-mehtyl-2-mercaptoimidazole on human thyroid iodine peroxidase. The mechanism of inhibition of human thyroid iodide peroxidase (TPO) by 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI) used in the therapy of hyperthyroid patients was studied in vitro. The inhibition of TPO by MMI was not restored either by dialysis or by dilution, but the inhibition by PTU was restored by both treatments. PTU interacted directly with the product of TPO action (oxidized iodide) in the reaction mixture without significantly affecting TPO activity. MMI interacted directly with TPO and inhibited enzyme activity, rather than interacting with the product (oxidized iodide). The inhibition was irreversible with MMI, but reversible with PTU. The concentrations of PTU and MMI producing 50% inhibition of TPO were 2 x 10-6m and 8 x 10-7m, respectively, 2-Mercaptoimidazole inhibited TPO reversibly but 1-methylimidazole and imidazole did not. Both the methyl and mercaptoresidues in MMI moiety are thought to be essential to its irreversible inhibition of TPO. The in vivo effect of MMI and PTU on TPO activity was also studied. TPO activities in the thyroid homogenate of rats to which MMI (2 mg per rat) or PTU (10 mg per rat) had been administered intraperitoneally were determined before and after dialysis  against buffer. TPO activity in the PTU treated thyroid homogenate was significantly lower than that in the control before dialysis, but the activity was restored to the control value after dialysis. On the contrary, TPO activity in the MMI treated thyroid homogenate was significantly lower than that in the control and was not affected by dialysis. These data may explain why MMI is a more potent inhibitor of iodination than PTU and may fit the clinical results observed when hyperthyroid patients are treated with these agents.


Integrated concentrations of luteinizing hormone and puberty. Integrated serum concentrations of luteinizing hormone have been compared among 30-minute collections from 10 boys (6-18 years old) and 5 girls (5-11 years old). This study suggests that perpubertal as well as pubertal boys have greater mean integrated concentrations of LH during sleep than during waking. One of two pubertal girls had greater concentrations of LH during sleep, while three prepubertal girls did not.


Secretory activity of isolated thyroid adenomas. The catabolic phase of thyroid secretion --thyroglubulin (Tg) uptake and hydrolysis, release of hormones--has been investigated in vitro, by incubating thyroid slices with 0.1% labelled Tg, in cases of isolated adenomas. Twenty-two specimens were examined: 11 cold follicular adenomas, 5 hot nodules from euthyroid patients, 2 untreated toxic adenomas, and 4 pretreated toxic adenomas. The results were compared with those obtained in 11 specimens of normal tissue. Tg pinocytosis (the amount of Tg taken up by the slices per mg of tissue) was severely impaired in all the non-toxic nodules, cold or hot, i.e., whatever the in vivo activity of the thyroid iodide pump. In toxic adenomas, every step of the catabolic hormonogenesis was activated: high pinocytotic activity, increased Tg hydrolysis, and the discharge of hormonal products; in pretreated cases, the whole process was slowed down. Enzymic activity of the acid hydrolases, beta-galactosidase and cathepsin, was elevated in all the nodules so far investigated, particularly in the toxic adenomas.


The effects of insulin on urinary urea and ammonia production. 15N-labelled glycine, ammonium chloride, and L-aspartic acid were infused intravenously into a series of non-diabetic adults at a constant rate for 4 to 8 hours. Midway during the infusion, a pulse of 8-10 units of regular insulin was injected as a pulse or infused at a constant rate for the remainder of the 15N infusion period. Frequent urine specimens were collected during the infusion period. The 15N enrichment of the urinary urea, ammonia, and amino acids increased smoothly with time until the insulin was given. The pulse of insuline caused a downturn on the N enrichment. We concluded from this that a) the effect of a pulse of insulin on human urea and ammonia metabolism lasted for about one hour; and b) insulin affected glutamine distribution in the plasma.


The role of 19-hydroxy-delta4-androstene-3,17-dione in the conversion of circulating delta4-androstene-3, 17-dione to estrone. A mixture of 4-14C-delta4-androstene-3, 17-dione and 6,7-3 H-19-hydroxy-delta4-androstene-3, 17-dione was intravenously injected into three women, and the 3H/14C ratios in urinary 19-hydroxyandrostenedione and urinary estrogens were determined. The ratios of 3H/14C in the estrogens were similar to those of the dose, while the ratios in urinary 19-hydroxyandrostenedione were mcuh higher than those of the dose. The fractional conversions of androstenedione to estrone and of 19-hydroxyandrostenedione to estrone are therefore similar. However, little, if any, 19-hydroxyandrostenedione produced during aromatization enters the circulation and mixes with injected 19-hydroxyandrostenedione.


Prolactin and testosterone: independent circulating levels in hyperprolactinemic and normoprolactinemic amenorrhea. The effect of prolactin suppression by bromocriptine. In order to elucidate the pituitary regulation of the female testosterone secretion, we studied by radioimmunoassay the circulating prolactin (PRL) and testosterone-dihydrotestosterone (T-dT) levels in 12 hyperprolactinemic and 12 normoprolactinemic patients with secondary amenorrhea. After the basal levels had been recorded, each patient was given bromocriptine for two weeks, 2.5 mg twice daily, and repeat estimations of the PRL and T-dT levels were done. We found no significant difference in the basal T-dT levels between normoprolactinemic and hyperprolactinemic patients, and no significant correlation between the PRL and T-dT levels in either group. Although the PRL levels of the hyperprolactinemic patients were greatly suppressed by bromocriptine, the T-dT levels showed no systematic change. In normoprolactinemic patients, the T-dT concentrations were somewhat lower during bromocriptine treatment, but the difference from basal levels was not statistically significant (0.05 less than P less than 0.1). Our results suggest that in patients with secondary amenorrhea PRL does not interfere directly with T-dT secretion, or vice versa.


Effect of norepinephrine infusion on plasma vasopressin levels in normal human subjects. Norepinephrine was continuously infused for 30 minutes at a rate of 0.2 and 0.3 mug/kg/min into 3 normal human subjects, and plasma vasopressin levels, plasma osmolality, hematocrit value, blood pressure, and heart rate were determined simultaneously. In norepinephrine infusion, an elevation of mean blood pressure and a decrease in heart rate was seen, and the degree of these changes was greater with the infusion at a rate of 0.3 mug/kg/min. Plasma vasopressin levels were suppressed by the infusion, and a dose-response relationship was recognized between the infusion at rates of 0.2 mug/kg/min and 0.3 mug/kg/min, while plasma osmolality did not change, and the hematocrit value rose slightly. These results suggest that norepinephrine-induced water diuresis is related to the suppression of the vasopressin release.


Insulin binding to monocytes and total mononuclear leukocytes from normal and diabetic patients. Isolated circulating mononuclear leukocyte preparations were obtained from 19 normal and 11 adult diabetic patients. No differences in monocyte, T cell, or B cell content were found when preparations from the two study groups were compared. The amount of insulin bound to cells from the diabetic patients was found to be decreased regardless of the cell type to which the data were normalized. This indicates that the previously reported decrease in the ability of mononuclear cells of diabetic patients to bind insulin was not a result of differences in mononuclear cell subpopulation.


Effect of carbidopa on prolactin, growth hormone and cortisol secretion in man. Serum prolactin, growth hormone and cortisol levels were examined in normal volunteers following administration of carbidopa, a peripheral decarboxylase inhibitor. 24-hour urinary tryptamine levels dropped markedly indicating that inhibition of decarboxylase was effective. Prolactin levels rose while growth hormone and cortisol remained unchanged. Since the tuberoinfundibular dopamine nerve terminals lie outside the blood brain barrier, this study suggests that these neurons are involved in prolactin but not in growth hormone or cortisol regulation. Findings are compatible with two alternate hypotheses--either that dopamine is a physiologic prolactin inhibiting factor (PIF) or that tuberoinfundibular dopamine neurons regulate the release of PIF.


Actions of growth hormone-release inhibiting hormone (somatostatin) on the renin aldosterone system. Administration of growth hormone-release inhibiting hormone (GH-RIH, somatostatin), as a 90 minute infusion (10 mug/min), to 3 healthy young men under conditons of active renin secretion acheived by pretreatment with furosemide (80 mg daily for 5 days), caused a mean 30% fall in plasma renin activity, which returned to basal levels immediately after stopping the GH-RIH infusion. Plasma aldosterone levels were not affected during the course of this experiment.


Uptake of 131I-19-iodocholesterol by an adrenal cortical carcinoma and its metastases. A case of a positive 131-I-19-iodocholesterol scan caused by a cortisol secreting adrenal carcinoma and its hepatic metastases is reported.


Plasma cortisol and cortisone in pregnancies with normal and anencephalic fetuses. Plasma cortisol (F), cortisone (E), and progesterone (P), were measured in the umbilical vein (UV), umiblical artery (UA), and maternal peripheral vein (MPV) of 17 normal patients, and of 8 patients carying anencephalic fetuses. The plasma F in MPV of patients undergoing vaginal delivery after labor of spontaneous onset was significantly higher than that of patients delivered by elective cesarean section, whereas the plasma F concentrations in the UA or UV of the 2 groups were not statistically different from each other. The anencephalic fetuses had UA plasma F and E concentrations which were significantly lower than those of normal fetuses, suggesting that a main portion of UA cortisol and cortisone originates in the fetal adrenal. The UV and MPV plasma F and E concentrations of patients carrying anencephalic fetuses did not differ, however, from those of normal patients, suggesting that these UV corticoids are derived mainly from maternal sources. The amniotic fluid cortisol levels of the patients carying anencephalic fetuses were lower than those observed in the normal pregnancies, suggesting that amniotic fluid cortisol is derived mainly from fetal sources.


Diagnosis and nomenclature of the disorders of the terminal portion of the aldosterone biosynthetic pathway. The abnormal steroid pattern in an inborn error in aldosterone biosynthesis consists of overproduction of glomerulosa zone 18-hydroxycorticosterone relative to aldosterone. The normal values for the excretory ratio of the major urniary metabolites of these two steroids are presented to provide a basis for the diagnosis of abnormalities in their ratio. The production of glomerulosa zone 18-hydroxycorticosterone relative to aldosterone is remarkably constant over a large range of absolute values, except in the disorder involving the terminal portion of the aldosterone biosynthetic pathway for which the term corticosterone methyl oxidase defect, Type 2, is suggested.


Reversible changes of the muscle cell in experimental phosphorus deficiency. Both animal and human studies suggest that either phosphorus depletion or hypophosphatemia might have an adverse effect on muscle function and composition. Recently a possible deleterious effect was noted in patients with chronic alcoholism. In this unexplained disease, a variety of toxic and nutritional disturbances could affect the muscle cell, thus obscuring the precise role of phosphorus. Accordingly, we examined eight conditioned dogs for the possibility that phosphorus deficiency per se might induce an abnormally low resting transmembrane electrical potential difference (Em) and alter the composition of the muscle cell. Eight conditioned dogs were fed a synthetic phosphorus-deficient but otherwise nutritionally adequate diet plus aluminum carbonate gel for a 28-day period followed by the same diet with phosphorus supplementation for an additional 28 days. Sequential measurements of Em and muscle composition were made at 0 and 28 days during depletion and again after phosphorus repletion. Serum inorganic phosphorus concentration (mg/100 ml) fell from 4.2 +/- 0.6 on day 0 t0 1.7 +/- 0.1 on day 28. Total muscle phosphorus content (mmol/100 g fat-free dry wt [FFDW]) fell from 28.5 +/- 1.8 on day 0 to 22.4 +/- 2.1 on day 28. During phosphorus depletion, average Em (-mV) fell from 92.6 +/- 4.2 to 77.9 +/- 4.1 mV (P less than 0.001). Muscle Na+ and Cl- content (meq/100 g FFDW) rose respectively from 11.8 +/- 3.2 to 17.2 +/- 2.8 (P less than 0.01) and from 8.4 +/- 1.4 to 12.7 +/- 2.0 (P less than 0.001). Total muscle water content rose from 331 +/- 12 to 353 +/- 20 g/100 FFDW (P less than 0.05). A slight, but nevertheless, significant drop in muscle potassium content, 43.7 +/- 2.0-39.7 +/- 2.2 meq/100 g FFDW (P less than 0.05) was also noted. After 4 wk of phosphorus repletion, all of these measurements returned toward control values. We conclude that moderate phosphorus depletion can induce reversible changes in skeletal muscle composition and transmembrane potential in the dog, and it apparently occurs independently of profound hypophosphatemia.


Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency. Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients...


The pathogenesis of coronary artery disease. A possible role for methionine metabolism. Homocystinuria, an abnormality of methionine metabolism is associated with severe vascular disease in infancy and childhood. Homocysteine is formed during the metabolism of methionine and accumulations of this and of cysteine-homocysteine mixed disulfide in the plasma indicate a partial block in the methionine degradation pathway. Methionine metabolism was investigated in 25 patients aged under 50 with angiographically proved coronary artery disease and in 22 control patients, of whom 17 had normal coronary arteries at angiography and 5 were healthy volunteers. After an overnight fast, venous blood was drawn before and 4 h after oral L-methionine, 100 mg/kg. Plasma methionine levels at 4 h were not different in the two groups, but there were significant differences in the levels of cysteine-homocysteine mixed disulfide. This was detected in 5 of 22 in the noncoronary group and in higher concentration in 17 of 25 coronary patients (P less than 0-01). Age, weight, height, body-mass index, glucose tolerance, fasting serum urate, and triglycerides were not different, but serum cholesterol was higher in the coronary patients (P lessthan 0.01). These results suggest a reduced ability to metabolise homocysteine in some patients with premature coronary artery disease when this pathway is stressed.


Insulin release is glucose anomeric specific in the human. The alpha-glucose anomer produces a greater insulin release than beta-glucose in various animal models. These glucose anomers were dissolved rapidly and administered intravenously to human volunteers at a high dose (0.5 g/kg) over a 3-min period or a low dose (3.5 g) over a 20-s period. Blood samples were obtained at frequent time intervals for measurement of whole blood glucose (ferricyanide), plasma glucose (beta-glucose oxidase) and serum immunoreactive insulin. The high-dose infusion test showed no differences between the anomers of either blood glucose or serum insulin levels. However, at the lower dose, the alpha-glucose anomer stimulated a significantly greater insulin release than did beta-glucose. It is concluded that the alpha-glucose anomer stimulates a greater insulin release than the beta-glucose anomer in human subjects at low but not at high doses intravenously and that this response is not apparently related to approximations of the degree of mutarotation. These results suggest that a steric specific glucose receptor site exists on the beta-cell as a rapid insulin release trigger, although the alpha-anomer does not exclusively produce this stimulation.


Inhibition of insulin degradation by nonsuppressible insulin-like activity. Nonsuppressible insulin-like activity, provided by three sources, was evaluated for its effect on the proteolytic degradation of insulin utilizing insulin protease obtained from rat liver homogenate as well as liver cell membranes. All three preparations of nonsuppressible insulin-like activity were found to be competitive inhibitors of insulin degradation. In addition human plasma was fractionated yielding an acetone precipitate which was found to have nonsuppressible insulin-like activity and to be a competitive inhibitor of insulin protease.


The induction of augmented granulocyte adherence by inflammation. Mediation by a plasma factor. The adherence of granylocytes to surfaces, measured in vitro in nylon fiber columns, is inhibited by in vivo administration of anti-inflammatory agents. Therefore, the effect of inflammation itself was assessed in blood from patients with acute inflammatory diseases. Mean adherence in these patients was twice normal (56.4 +/- 5.6% vs. 29.4 +/- 5.2%); their plasma contained a factor that augmented adherence of normal cells to 47.5 +/- 5.6% whereas the patient's cells showed a normal level of adherence (34.0 +/- 6.8%) when resuspended in normal plasma. Although exudate fluid from exprimental inflammation also contained the augmenting factor, cells from the exudate maintained their high level of adherence after washing and suspension in normal plasma. The augmenting factor detected in plasma from patients with inflammation was not present in serum and was inactivated by heating plasma to 56 degrees C for 30 min; restoration of augmenting activity was accomplished by addition of 20% guinea pig serum to the heat-treated plasma. Because the guinea pig serum itself did not increase adherence when added to normal plasma, it appears that the augmenting factor is heat-stable, but requires a heat-labile cofactor like complement. Sephadex G-200 fractionation of inflammatory plasma showed adherence-augmenting activity in the majority of fractions, with peak activity in the fractions corresponding to approximate molecular wts of 30,000, 160,000 and 400,000.


Effect of renal sympathetic nerve stimulation on proximal water and sodium reabsorption. The renal responses to sympathetic nerve stimulation were studied in saline-expanded rats. The left kidney was partially denervated by crushing the left greater splanchnic nerve. Then the distal portion of the nerve was stimulated with square wave pulses of 0.5 ms duration, voltage twice threshold, and 1 or 2 Hz frequency while monitoring the compound action potential. Fibers with conduction speeds of 13-17 m-s-1 and of 0.7-1 m-s-1 were identified. Only stimulation of the latter appeared to produce changes in renal Na and water excretion. Whole kidney and individual nephron studies were performed alternating control and nerve stimulation periods. Nerve stimulation produced approximately a 25% reduction of the left kidney urine volume and sodium excretion. Glomerular filtration rate and renal plasma flow remained unchanged. Right kidney Na and water excretion, glomerular filtration rate, and renal plasma flow remained constant. In the left kidney, during nerve stimulation, the tubular fluid to plasma inulin concentration ratio increased significantly in the late proximal tubule. We conclude that the antidiuresis and antinatriuresis seen during sympathetic nerve stimulation were caused by increased sodium and water reabsorption in the proximal tubule, probably mediated by the stimulation of slowly conducting unmyelinated fibers. These responses appeared to be unrelated to systemic or intrarenal hemodynamic changes.


Intrinsic differences in various segments of the proximal convoluted tubule. Until recently it has not been possible to compare directly the function of superficial and juxtamedullary nephrons. The present studies, using in vitro microperfusion, were designed to examine whether functional differences exist between proximal convoluted tubule segments of superficial and juxtamedullary nephrons. Electrophysiological studies showed that major differences exist between the relative chloride and sodium permeabilities of these segments. In the 1st mm of the superficial proximal convoluted tubule, the permeability to sodium was greater than that to chloride, whereas in the 2nd mm of the superficial proximal convoluted tubule and all later segments, the permeability to chloride was greater than that to sodium. The juxtamedullary proximal convoluted tubule was found to differ from the superficial proximal convoluted tubule in two respects: first, the relative permeabilities to chloride and sodium did not differ in the various segments of the juxtamedullary proximal convoluted tubule; second, the permeability to sodium was greater than to chloride throughout. When perfused with a solution lacking glucose and amino acids, the superficial and juxtamedullary convolutions exhibited the same transepithelial potential change, a reversible decrease to less than -- 1 mV. It thus appears that in both convolutions there exists electrogenic sodium transport coupled to the transport of these organic solutes. This differs from pars recta of both of these nephrons, which have been shown to exhibit electrogenic sodium transport independent of organic solutes. However, when perfused with a solution lacking glucose and amino acids but also containing high chloride and low bicarbonate concentrations, the superficial convolution developed a significantly more positive potential than the juxtamedullary. This difference reflects greater relative chloride permeability in the superficial proximal convolution. These studies show that intrinsic functional differences exist between proximal convoluted tubules obtained from the superficial and juxtamedullary nephron populations.


Human bone marrow lymphocytes. Cytotoxic effector cells in the bone marrow of normal individuals. This study was undertaken to determine the capability of lymphocytes in the bone marrow of normal individuals to mediate nonspecific killer cell functions in assays of phytohemagglutinin (PHA)-induced cellular cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) against 51Cr-labeled chicken erythrocyte target cells. Relatively pure mononuclear cell suspensions were obtained from bone marrow aspirates in 30 normal volunteers by sucrose gradient centrifugations and from the peripheral blood of the same individuals by Hypaque-Ficoll density centrifugations. At an effector: target ratio of 10:1, the PHA-induced cellular cytotoxicity of peripheral blood was 78.8 +/- 1.3%, while that of bone marrow was not significantly less at 66 +/- 9% (P greater than 0.1). At low effector:target ratios, the ADCC of bone marrow was negligible, while at higher effector:target ratios (20:1) bone marrow ADCC was 69 +/- 3.7%, which was comparable to that of peripheral blood. The lymphocytes themselves in the mononuclear cell suspensions of both peripheral blood and bone marrow were capable of cytotoxicity activity since depletion of monocytes from the suspensions by adherence to rayon wool and G-10 Sephadex columns did not remove the cytotoxic activity. Blocking of the Fc receptor on the effector cells by the addition of aggregated gamma globulin to the cultures suppressed the ADCC but not the PHA-induced cellular cytotoxicity of both peripheral blood and bone marrow, indicating that ADCC is dependent on an Fc receptor on the effector cell in both compartments. These studies demonstrate that the bone marrow of normal humans contains populations of lymphoid cells which have highly efficient killer cell capacities. It is uncertain what portion of these cells arise in the bone marrow and what portion enter the bone marrow parenchyma as part of the recirculating lymphocyte pool. These findings have relevance in the clearer understanding of the killer cell potential of grafted human marrow, as well as the bone marrow sequestration of functionally capable lymphocyte subpopulations in disease states and during chemotherapy.


The relationship of structure and function in human Hageman factor. The association of enzymatic and binding activities with separate regions of the molecule. Three regions of the human Hageman factor molecule termed the c, d, and e regions have been defined. Division of the molecule into these three regions is based on the analysis of fragments obtained by enzymatic cleavage during fluid-phase activation. The three regions have the following properties: (a) the c region has a mol wt of 40,000, has the capacity to bind to negatively charged surfaces, and does not have detectable enzymatic activity; (b) the e region possess a mol wt of 28,000 has enzymatic activity, and does not bind to negatively charged surfaces; (c) the d region has a mol wt of 12,000, is located between the c and e fragments but has not been detected as a freely existing polypeptide, and can bind firmly to negatively charged surfaces. The preparation of antibodies specific for the c and e regions is described as well as their use in defining the electrophoretic characteristics of the cde, cd, de, c, and e polypeptide fragments of Hageman factor. Evidence is given showing that the e region, but not the c or d, is released from a negatively charged surface when bound Hageman factor is exposed to proteolytic enzymes or whole plasma and that when this occurs in the presence of normal plasma, the e fragment becomes bound to C1 esterase inhibitor.


Jejunal and ileal absorption in patients with chronic renal disease. Effect of 1alpha-hydroxycholecalciferol. Calcium absorption in 30-cm segments of small intestine was measured by constant perfusion of test solutions containing different concentrations of calcium gluconate. In both the jejunum and ileum, calcium absorption rates increased progressively as luminal calcium concentration was increased stepwise between 1 and 20 mM. Although calcium transport was not saturable within these limits, unidirectional flux ratios of calcium (47Ca) suggest that calcium absorption is active in both the jejunum and ileum. Calcium absorption in patients with chronic renal disease was markedly depressed in both regions of the small intestine. This was due to decreased flux out of the lumen; flux in the reverse direction was normal. Flux ratios in the renal disease patients showed no evidence for active calcium transport. Treatment of these patients for 1 wk within 2 mug/day of 1alpha-hydroxycholecalciferol [1alpha-(OH)-D3] restored net calcium absorption and unidirectional calcium flux out of the lumen to normal values in the jejunum; in the ileum, 1alpha-(OH)-D3 increased calcium absorption 60-83% of normal at the various luminal calcium concentrations. 1alpha(OH)-D3 had no effect on unidirectional calcium flux into the lumen or on xylose and electrolyte absorption in either area of the small intestine.


Influence of dietary phosphorus on renal phosphate reabsorption in the parathyroidectomized rat. Inorganic phosphate (Pi) reabsorption was studied during Pi infusion, after acute or chronic thyroparathyroidectomy (TPTX), in rats stabilized on a high-phosphorus (1% P) or a low-phosphorus (0.02% P) diet. After acute TPTX, there were no consistent differences in Pi reabsorption between the high- and low-phosphorus dietary groups. After chronic TPTX, the rats stabilized on the low-phosphorus diet exhibited nearly complete Pi reabsorption at every plasma Pi level, while the animals receiving the high-phosphorus diet manifested a marked phosphaturic response to Pi infusion. In addition, Pi reabsorption was significantly increased in the chronic TPTX low-phosphorus rats which achieved the highest filtered Pi loads, while their urine remained essentially phosphate-free. Dietary phosphorus-dependent alterations in Pi reabsorption may play a significant role in establishing the rate of Pi excretion per nephron under certain circumstances and should be considered in the interpretation of studies investigating renal Pi handling. The ability of phosphorus-depleted animals to maintain a phosphate-free urine during Pi loading would favor the rapid repletion of body phosphorus stores.


Mechanism of production of intestinal secretion by elevated venous pressure. A study was carried out to elucidate the physiological mechanisms responsible for the intestinal secretion produced by venous pressure elevation. In dogs, measurements were made of the rate and composition of small intestinal secretion, rate of flow and composition of intestinal lymph, plasma composition, and mucosal water content, all in response to elevations of intestinal venous pressure. Venous pressure elevations above a threshold value of 30-35 cm H2O produce secretion at a rate of approximately proportional to the value of the pressure minus the threshold value. Above the threshold value, there were large increases in the rates of lymph flow and net sustained transcapillary filtration. These rates were also roughly proportional to the incremental venous pressure. It is concluded that intestinal secretion produced by elevated venous pressure is almost surely secretory filtration, a passive process with the driving force for secretion an increase in mucosal tissue fluid pressures to values of only some 4-6 cm H2O. The increased tissue fluid pressure not only provides the driving force but also produces an increase in the hydraulic permeability of the epithelium without which the driving force would be ineffective. The transepithelial channels are large enough to permit insulin to pass freely and even plasma protein to pass in large amounts, and hence are most probably intercellular. Secretory filtration probably represents a general pathophysiological response of transporting epithelia to elevated tissue fluid pressure. It is proposed that the threshold value for secretion and associated changes is explained by dilution of the tissue fluid protein colloid osmotic pressure in a small subepithelial, juxtacapillary compartment.


The effect of changes in perfusion pressure on uteroplacental blood flow in the pregnant rabbit. The effect of perfusion pressure on uteroplacental blood flow was determined in pregnant rabbits utilizing the radioactive microsphere method. Control mean arterial pressure, 93 mm Hg +/- 2.6 SEM, was raised by carotid ligation to 109 +/- 4.1 mm Hg and then reduced with antihypertensive drugs to 74 +/- 1.3 mm Hg. Over this range of pressure there was no significant change in cardiac output, 605 +/- 36, 523 +/- 37, and 540 +/- 39 ml/min; or uteroplacental blood flow, 30 +/- 3.2, 27 +/- 5.2, and 29 +/- 4.5 ml/min, respectively. When prostaglandin synthesis was inhibited with either indomethacin or meclofenamate (2 mg/kg), uterine vascular resistance was higher but maintenance of uteroplacental flow occurred over a perfusion pressure of 89 +/- 6.7-115 +/- 9.3 mm Hg. With more severe hypotension induced with trimethaphan, control arterial pressure fell from 92 +/- 2.4 to 39 +/- 0.9 mm Hg, cardiac output fell from 514 +/- 17 to 407 +/- 22 ml/min (P less than 0.025) and uteroplacental blood flow fell from 6.1 +/- 0.9 to 2.5 +/- 0.9% of cardiac output (P less than 0.05), which represented an absolute fall from 32.4 +/- 5 to 10.6 +/- 3 ml/min (P less than 0.025). There was no significant change in renal blood flow expressed as percentage of cardiac output, 14.9 +/- 2 and 13 +/- 1.5%, or in absolute flow, 75 +/- 7.7 and 54 +/- 7 ml/min with trimethaphan-induced hypotension. These studies indicate that uteroplacental blood flow is maintained relatively constant over a range of perfusion pressure of 60-140 mm Hg in both normal and prostaglandin-inhibited pregnant rabbits. However, with reduction in pressure to 36-42 mm Hg, uteroplacental blood flow falls, expressed as a percentage of cardiac output and in absolute flow.


Leukocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions. Because of the many potent biological capabilities of the blood granulocytes, and their contact with platelets in various physiologic and pathologic states, a possible interaction between granulocytes and platelets was investigated. Platelets were purified by gel filtration and via a dialysis membrane were separated from suspensions of autologous granulocytes prepared by dextran sedimentation and resuspended in modified Tyrode's buffer. After 20 min at 37 degrees C platelet aggregation was shown to be diminished by such exposure, as compared to the aggregation of platelets incubated with dialysates of buffer only. When granulocytes were stimulated by the addition of 1.1-muM latex spheres as target particles for phagocytes, the dialysate of these cells exhibited greatly enhanced platelet-inhibitory properties. The addition of catalase to the platelets abolished the effect of exposing these cells to the dialysate of resting granulocytes and markedly inhibited the effect of exposing the platelets to the dialysate of phagocytosing granulocytes. Catalase treated with 3-amino-1,2,4-triazole had no platelet-protective capacity. Purified suspensions of lymphocytes released no platelet-inhibitory principle under these experimental conditions. Hydrogen peroxide in the dialysate of granulocytes was measured directly with an assay involving an H2O2-induced decrease in the fluorescence of scopoletin catalyzed by horseradish peroxidase. The dialysate of phagocytosing granulocytes contained 0.86 +/- 0.55 nmol H2O2/2.5 X 10(7) granulocytes when sampled at 20 min. By an alternate measurement technique in which scopoletin and horseradish peroxidase were present in the dialysate from time zero, the mean amount of H2O2 in the dialysate reached 4.0 +/- 1.3 nmol/2.5 x 10(7) granulocytes at 20 min. This discrepancy suggested the consumption of H2O2, possibly mediated by the granulocytes themselves. This possibility was investigated by the addition of exogenous H2O2 to the test system. Both granulocytes and platelets enhanced the disappearance of H2O2 from the dialysate, and the amount consumed was proportional to the amount of H2O2 added to the system. Glucose oxidase at 12 M U/ml plus glucose in excess resulted in the production of H2O2 at a rate and final amount comparable to that produced by phagocytosing granulocytes. This mixture, when substituted for phagocytosing granulocytes in the standard dialysis membrane experiment, induced an inhibition of platelet aggregation similar to that caused by the granulocytes. The observation that the release of H2O2 by the blood granulocyte influences platelet function suggests a potential role for the granulocyte in the regulation of hemostasis or thrombosis.


Mechanism of the ventilatory response to carbon monoxide. The effects of carbon monoxide on ventilation were studied in unanesthetized goats. Responses to single breaths of 10-25% CO in O2, which rapidly raised carboxyhemoglobin (COHb) from 5 to 60%, were considered to reflect peripheral chemoreceptor-mediated reflexes whereas responses to continuous inhalation of 1% CO in O2, which slowly raised COHb from 0 to 60%, were considered to reflect both peripheral chemoreceptor and nonperipheral chemoreceptor mechanisms. In each of six goats, single breaths of CO failed to elicit any immediate ventilatory response. However, slow buildup of carboxyhemoglobinemia in the same animals always elicited ventilatory stimulation (from a mean of 7.43 to 16.02 liter/min, P less than 0.001) beginning 5-6 min after onset of 1% CO in O2 inhalation when COHb saturation reached 50-60%. In eight studies of six animals HCO3- concentration fell (from 21.3 to 15.8 meq/liter; P less than 0.001) and lactate concentration rose (from 2.5 to 4.2 meq/liter; P less than 0.05) in the cisternal cerebrospinal fluid during the CO-induced hyperpnea. Additional studies ruled out ventilatory stimulation from left heart failure or enhanced chemo-sensitivity to carbon dioxide. Although the delayed hyperpnea was associated with a hyperdynamic cardiovascular response to CO, blockade of these circulatory effects with propranolol (2 mg/kg) failed to abolish the delayed hyperpnea; however, the propranolol did unmask an element of ventilatory depression which preceded the hyperpnea. Conclusions were: (a) hyperventilation in response to CO inhalation is not mediated by the carotid bodies; (b) the delayed hyperpnea in response to CO inhalation is primarily due to brain-cerebrospinal fluid acidosis; (c) mobilization of body CO2 stores due to the circulatory response to CO may obscure an initial depression of ventilation by CO.


Effect of protein ingestion on splanchnic and leg metabolism in normal man and in patients with diabetes mellitus. The inter-organ flux of substrates after a protein-rich meal was studied in seven healthy subjects and in eight patients, with diabetes mellitus. Arterial concentrations as well as leg and splanchnic exchange of amino acids, carbohydrate substrates, free fatty acids (FFA), and ketone bodies were examined in the basal state and for 3 h after the ingestion of lean beef (3 g/kg body wt). Insulin was withheld for 24 h before the study in the diabetic patients. In the normal subjects, after protein ingestion, there was a large amino acid release from the splanchnic bed predominantly involving the branched chain amino acids. Valine, isoleucine, and leucine accounted together for more than half of total splanchnic amino acid output. Large increments were seen in the arterial concentrations of the branched chain amino acids (100-200%) and to a smaller extent for other amino acids. Leg exchange of most amino acids reverted from a basal net outut to a net uptake after protein feeding which was most marked for the branched chain amino acids. The latter accounted for more than half of total peripheral amino acid uptake...


Variability of 24-hour urinary creatinine excretion by normal subjects. Creatinine excretion was studied in eight healthy males who collected 54 to 97 24-hour urine specimens. Significant differences among subjects in mean creatinine excretion were only partly explained by differences in body weight and surface area. Considerable daily within-subject variation in creatinine excretion during normal activity was found. Standard deviations were from 10.5 to 14.4 per cent of the mean, and ranges varied from 50 to 79 per cent of the mean. Day-to-day variation appeared to be time dependent rather than entirely random, and could not be explained by unreliability of the assay technique or by incomplete collections. Creatinine excretion values were normally distributed in seven of eight subjects. Individual variation from day to day limits the value of urinary creatinine excretion as an index of the completeness of 24-hour urine collections.


Renal clearance of theophylline in man.

Renal clearance of theophylline in man. The renal clearance of theophylline was determined in 12 experiments with six healthy human adult volunteers and was found to decrease from 8.50+/-2.20 to 276.+/-1.13 ml/min/m2 during 8 hours after oral administration of a single dose of theophylline, 200 mg/m2. There was a strong and highly statistically significant positive correlation between the renal clearance of theophylline and the urine flow rate for each subject, but there was no consistent correlation between renal clearance and plasma concentrations of theophylline.


Relationship of sulfamethazine disposition kinetics to acetylator phenotype in man. A preliminary study. The relationship between sulfamethazine disposition kinetics and acetylation phenotype was studied in man. Sulfamethazine pharmacokinetic parameters were determined after the administration of the drug as an oral suspension. When the half-life, acetylation rate constant, or per cent available dose excreted in the urine as acetylsulfamethazine of each subject was plotted on frequency distribution histograms, bimodal distribution patterns were observed. However, when acetylation clearance values were plotted in the same manner, an apparent trimodal pattern was seen. The failure to identify the presumed homozygous rapid acetylator using the commonly employed indices of drug metabolism, i.e., half-life, metabolic rate constant, or per cent of the dose metabolized, was attributed to a significant increase in the apparent volume of distribution of this genotype, as well as the low renal clearance of sulfamethazine found in all genotypes. This preliminary study points out the value of using metabolic clearance as an index of drug metabolizing capacity and suggests its application to further pharmacogenetic studies.


Plasma protein binding of diazepam and tolbutamide in chronic alcoholics. The increased incidence of drowsiness in hypoalbuminemic patients administered diazepam and more rapid clearance of tolbutamide in cirrhotics may be due to changes in plasma protein binding. The binding of diazepam and tolbutamide was studied by equilibrium dialysis at 37degreesC over a total drug concentration range of 1 to 10 mug/ml and 50 to 300 mug/ml, respectively, in plasma from 21 normal and 14 alcoholic subjects. At 1 mug/ml, diazepam plasma protein binding (+/- S.D.) was 98.5+/-0.4 per cent in normals and 97.8+/-1.2 per cent in alcoholics; at 100 mug/ml, tolbutamide binding was 97.8+/-0.3 per cent in normals and 95.1+/-4.2 per cent in alcoholics. For both agents at all concentrations, the binding to plasma from alcoholics was significantly decreased (P less than 0.01-less than 0.02). The extent of binding of both drugs was dependent on the albumin concentration. These findings suggest that important changes in pharmacologic effect, distribution, and clearance of diazepam and tolbutamide can be anticipated in alcoholics with hypoalbuminemia.


A long-term study of low-dose aerosolized terbutaline sulfate. The purpose of this study was to evaluate the safety and efficacy of a new lowdose terbutaline sulfate aerosol (375 micrograms) over a six-week period. The question of drug tolerance was also evaluated. Twenty-four ambulant patients with chronic obstructive pulmonary disease with a component of reversible airway disease was evaluated. The patients were tested at two-week intervals during a six-week period. The patients abstained from all bronchodilatory medications for at least 10 hours prior to the time of evaluation. The evaluation consisted of baseline FEV1 and MMEFR determinations, ECG, CBC, urinalysis, and renal and liver function tests. After the terbutaline was administered, a rhythm strip and pulmonary function tests were repeated at 5, 15, 30, 60, 120, and 180 minutes. Throughout the six-week study there was a statistically significant increase in FEV1 and MMEFR, (P less than 0.001). No change was noted in the systolic blood pressures; however, heart rate and diastolic blood pressure decreased significantly. No drug tolerance, paradoxical bronchospasm, subjective side effects, no abnormal laboratory results were noted during the study period.


Oral terbutaline sulfate--amelioration of exercise-induced bronchospasm. Bronchospasm can be induced in asthmatics when exercised according to a multistage branching treadmill protocol that allows them to achieve 80 per cent of their age-predicted maximal heart rate. This present study was undertaken to investigate the effect of oral terbutaline sulfate in ameliorating exercise-induced bronchospasm. Ten asthmatics were exercised, and FEV1.0 and MMEFR were assessed while standing, using a Jones Pulmonar II waterless spirometer at 5, 15, and 30 minutes after exercise. Three hours before exercise, the subjects received no medication, oral placebo, or 5 mg terbutaline sulfate orally. At all time intervals after exercise, the pulmonary function of the group pretreated with oral terbutaline was superior when compared to the no-medication group (P less than 0.01) and to the placebo-treated group (P less than 0.05). Oral terbutaline appeared to normalize the exercise tolerance of the asthmatics and restore physiologic pulmonary airway conductance by the parameters of FEV1.0 and MMEFR.


Therapeutic efficacy of a new anhydrous theophylline dosage form. A clinical study was performed on 41 asthmatic patients. After complete physical and spirometric examinations, they received capsules containing 200 mg anhydrous theophylline per capsule t.i.d. for three weeks. All other antiasthmatic medications were omitted during this study. The results showed that these capsules (Elixophyllin Capsules) produced statistically significant improvement in wheezing, dyspnea, global clinical assessment, vital capacity, and forced expiratory volume at 1 second. Side effects, which were generally mild and transient, were those usually reported for theophylline and were observed in 11 of the 41 patients. One patient dropped out of the study because of extreme nausea. It was concluded that theophylline as a single entity can provide significant improvement in asthmatic patients at a total daily dose of 600 mg.


Immunologic comparison of phospholipases A present in Hymenoptera insect venoms. A comparative study was made of the effect of anti-bee venom phospholipase A2 serum and anti-bald face hornet venom serum on the activity of phospholipase A2 present in yellow hornet, yellow jacket, bald face hornet, and honeybee venoms. Activity of phospholipase A2 present in the venoms was measured titrimetrically using egg yolk dispersion as the substrate. Antiserum against pure bee venom phospholipase A2 activity completely suppressed bee venom phospholipase A2 activity but failed to inhibit phospholipase A2 activity in yellow jacket, yellow hornet, and bald face hornet venoms. Anti-bald face hornet venom serum suppressed the activity of phospholipase A2 present in the four insect venoms with the inhibitory effect decreasing in the order: bald face hornet greater than yellow hornet greater than yellow jacket greater than honeybee. A combination of immunoelectrophoresis and immunodiffusion techniques gave indication of common antigenic sites in a component of yellow hornet venom and a component of bald face hornet venom. The results suggest that IgG antibodies against pure bee venom phospholipase A2 may not give any protection against the reaction(s) due to the phospholipase A2 in yellow hornet, yellow jacket, and bald face hornet venoms, while antibodies against bald face hornet venom phospholipase A2 may give some cross-protection.


Imported fire ant hypersensitivity. Studies of human reactions to fire ant venom. It is now apparent that venom and venom components of the Hymenoptera superfamilies of Apida (honeybee) and Vespida (wasps, yellow jackets, and hornets) are becoming increasingly important in the diagnosis and treatment of hypersensitivity reactions. Stings from fire ants (superfamily Formicidae, family Myrmicinae) have also been recognized as causes of systemic reactions in man. Fire ant venom is unique in its composition, consisting mainly of alkaloids in aqueous suspension with only trace amounts of protein. This study compares the skin reactivity of fire ant venom and synthesized alkaloid components with the whole body extract (WBE) of fire ants and other Hymenoptera. The venom as well as the WBE of fire ants was found useful for skin test diagnosis of sensitive individuals. There appear to be cross-reactive or shared antigens between fire ant venom, WBE, and WBE of other Hymenoptera. Successful passive transfer of skin reactivity to nonsensitive individuals was accomplished with sera from sensitive individuals. Loss of this passive transfer by heating sera at 56 degrees C for 4 hr is evidence in favor of IgE mediating the positive skin test to the venom.


The effect of cholinergic agents on a canine model of allergic asthma. Pharmacologic agents capable of influencing the activity of the cholinergic nervous system were evaluated for possible effects on a canine model of allergic asthma. Dogs with an active cutaneous sensitivity to an antigen (Ag) prepared from Ascaris suum were subjected to repeated aerosol Ag provocations at 2-wk intervals. Ag-induced increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance (Cdyn) were obtained in the presence and absence of drugs. The intravenous administration of atropine or chlorisondamine before, or atropine after, Ag-provocation, in most cases did not result in a significant reduction in Rp increases; however, in some animals an attenuation of the Rp increase was apparent. Aerosol administration of atropine or physostigmine prior to Ag provocation was without effect on Rp increases. None of the agents studied appeared to effect Ag-induced decrease in Cdyn. Similarly, intravenous atropine produced a slight antagonism of exogenous histamine-induced increases in Rp and decreases in Cdyn. These results suggest that the cholinergic nervous system is neither a prominent nor a consistent factor in Ag-induced increases in Rp in dogs.


Analysis of the potency of extracts of June grass pollen by their inhibitory capacities in the radioallergosorbent test. The potencies of 11 commercial extracts of June grass pollen were analyzed by skin test end point titrations and compared to potencies as determined in vitro (1) by the radioallergosorbent test (RAST), (2) by Group I antigen content, and (3) by protein nitrogen units (PNU). RAST potencies were determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen, and they were expressed as the quantity of extract required for 50% inhibition of binding. Potencies determined by skin testing in 8 patients were significantly related among the various patients in 19 of 27 comparisons and showed differences of up to 95,000-fold in the strengths of the extracts. Estimation of potencies by RAST inhibition showed approximately a 100-fold difference among the extracts and in 5 of 8 cases these were significantly related to potencies measured by skin tests. Similarly, PNU determinations and Group I determinations were also significantly related to potencies by skin test titration in 5 of 8 and in 4 of 8 comparisons, respectively. Comparison of the geometric mean skin test potencies with RAST, PNU, and Group I potencies revealed that all were significantly related to skin test potencies although the correlation of RAST and skin potency was the highest. The results indicate that measurement of potency by RAST inhibition compares favorably with other in vitro measurements of potency. These results are compared with those of a prior study with extracts of short ragweed, and the reasons for the differences between the results in the two studies are discussed.


The stability of short ragweed pollen extract as measured by skin test and antigen E. A short ragweed pollen extract stored at 4 degrees C was shown to retain its skin reactivity for 18 mo, although 60% of the antigen E was lost. When stored at higher temperatures, the extracts had a markedly reduced skin reactivity for some individuals, but retained full reactivity in others even though the antigen E was markedly diminished. Thus, some ragweed-sensitive individuals are very sensitive to antigen E, which is heat-labile, but some have a major sensitivity to other allergens, which are heat-stable. During the 18-mo course of this study, skin test reactions to a similar extract did not show significant variability.


Factors which influence late cutaneous allergic responses. Increased doses of ragweed antigen E, grass pollen extract, and the IgG of sheep antiserum to human IgE (anti-IgE) were injected in intracutaneous tests in humans. With increasing doses of reactants, in individual subjects, there were the expected increases in the sizes of early wheal-and-flare responses; moreover, there was an associated increase in the magnitude and the frequency of late cutaneous allergic responses (LCAR). LCAR were shown to be a usual sequel to allergic reactions of sufficient intensity. In persons with allergic bronchopulmonary aspergillosis, cutaneous responses to Aspergillus fumigatus allergen were compared with responses to other allergens and to anti-IgE; in relation to early wheal-and-flare responses, the corresponding LCAR induced by A. fumigatus were approximately the same size as those induced by the other reactants. Skin reactions induced by the prick test method and by intracutaneous injection were compared; for early responses of given sizes, there were apparently equal tendencies to proceed to LCAR in reactions to allergen in the two types of administration. In one subject, anti-IgE-induced responses in the skin of a forearm with sensory denervation were compared with responses in the other normally innervated forearm. At the denervated site was the expected absence of the early flare but no apparent reduction in LCAR. Similarly, local anesthesia immediately prior to anti-IgE eliminated the flare of the early response but did not prevent the occurrence of a LCAR.


Passive transfer of experimental hypersensitivity pneumonitis with lymphoid cells in the rabbit. Although precipitating antibody is associated with human hypersensitivity pneumonitis, there is evidence that cell-mediated hypersensitivity may be involved in disease pathogenesis. In this study, interstitial, and peribronchial lesions were produced by respiratory challenge of rabbits passively sensitized with ovalbumin-sensitive lymph node cells. Ovalbumin sensitivity of donor rabbits and lymph node cells was demonstrated by skin testing, migration inhibition factor (MIF) production using alveolar wash cells as migrating cells, and lymphocyte stimulation. Passive cell transfer was accomplished by intraperitoneal injection with all lymph node cells obtained from one donor transferred to one recipient. Recipients were challenged by aerosol or intratracheal injection of antigen immediately or 24 hr after passive sensitization and were killed 48 hr after challenge. Lesions in rabbits passively sensitized by lymph node cells and challenged with antigen by intratracheal inoculation consisted of focal pneumonitis with intra-alveolar edema and infiltrates of mononuclear cells in alveoli and alveoli septa. Aerosol challenge of passively sensitized animals produced similar changes, but peribronchial tissue containing macrophages and germinal centers was prominent in this group. Antiovalbumin serum recipients challenged by intratracheal injection demonstrated only mild peribronchial mononuclear cell infiltrates, without pneumonitis. Control animals receiving lymph node cells only or challenge only demonstrated no changes in lung histology.


Intranasal immunization with attenuated live influenza vaccine in asthma. Attenuated live intranasal influenza vaccine ("Alice") was given to 20 asthmatics and 9 control subjects. Pulmonary function were performed before and after, with emphasis on tests of small airways function (using flow volume curves with air and a helium-oxygen mixture). In subjects with a low influenza A antibody titer, there was a 4-fold rise in titer to the vaccine, whereas those subjects with a high titer showed no rise. There were no significant changes in pulmonary function in any parameters measured, and no significant symptoms were reported. We have concluded from this study that "Alice" appears safe for use in asthma and was capable of producing an antibody titer rise in persons with low titers.


Clinical and bronchial provocation studies in patients with meatwrappers' asthma. Reactive airway disease has been meatwrappers employing polyvinyl chloride (PVC) soft wrap film and thermally activated price labels. Sixty-nine percent of 96 meatwrappers responding to a mail survey had work-related respiratory, mucous membrane, or systemic complaints. Smokers developed more pronounced symptoms than nonsmokers. Work-simulated bronchial provocation tests were carried out in 14 symptomatic meatwrappers. Following a 3-hr exposure to polyvinyl chloride fumes, 3 of 11 workers developed a mean decrease of 25% in FEV, and 7 of 11 workers developed a mean decrease of 13 mm Hg in their PaO2. Following a 30-sec to 20-min exposure to price label adhesive fumes, 9 of13 workers developed a mean decrease of 49% in FEV1 and 12 of 13 developed a mean decrease of 20 mm Hg in their PaO2. Though a hypersensitivity reaction to one or more components in PVC or price label adhesive emissions is suspected, the precise pathogenesis of this syndrome remains unknown. The entire spectrum of meatwrappers' syndrome must be interpreted as a complex response to both PVC and price label adhesive fume exposure.