Showing papers by "Medical University of South Carolina published in 1978"

Scanning and transmission electron microscopic study of Escherichia coli O15 (RDEC-1) enteric infection in rabbits

Abstract: RDEC-1 is a piliated strain of Escherichia coli that was isolated from and produces diarrhea in rabbits without invading the mucosa or synthesizing one of the classical enterotoxins. Previous histological and fluorescent-antibody studies of RDEC-1 diarrhea revealed an acute inflammatory response and large numbers of RDEC-1 associated with (adhering to) the mucosal surface of the ileum, cecum, and colon. The purpose of the present investigation was to further elucidate the histopathology by scanning (SEM) and transmission (TEM) electron microscopy. SEM revealed aggregates of bacteria on the surface of the gut; their distribution was patchy in the ileum and diffuse in the cecum and colon. Bacteria were in contact with each other and appeared to be closely associated with the epithelial surface. TEM showed that the brush border region of the epithelial cells was found to be in varying stages of degeneration, and the bacteria could not be seen adhering to the mucosal cells unless the brush border was absent. Bacteria were in close contact only with epithelial cells that had lost their brush border. The space between the bacteria and the epithelial cells was 11 nm, and it appeared to be filled, in most cases, with densely stained material. This E. coli rarely penetrated epithelial cells, but when it did; it was found in the supranuclear region and never reached the lamina propria. From previous and present studies, it seems probable that RDEC-1 produces diarrhea in rabbits by a mechanism that may be cytotoxic and differs from the classic mechanisms by which E. coli produces diarrhea.

The predictable relationship between plasma levels and dose during chronic propranolol therapy

Abstract: The present study has examined the relationship between plasma propranolol concentrations and dose during chronic propranolol therapy and the between‐patient variation in this relationship under rigorously controlled conditions. Peak (2 hr) and trough (6 hr) plasma concentrations were measured at carefully established steady‐state conditions in 46 patients with hypertension or coronary artery disease. All patients were hospitalized in a clinical research unit. Propranolol doses ranged from 40 to 960 mg/day (every 6 hr). Propranolol was measured by gas chromatography‐mass spectrometry using a stable isotope‐labeled internal standard. Peak plasma propranolol (ng/ml) was linearly related to dose over the range 160 to 960 mg (y = 1.11x − 111; correlation coefficient = 0.96); a non linear relationship exists over the range 40 to 160 mg. Trough plasma concentrations were 51 ± 9% (mean ± SD) of the peak concentrations over the entire dose range. Between‐patient variation in plasma propranolol was much smaller than has previously been reported in spite of the fact that the patient population studied was quite heterogeneous and that numerous other drugs were concomitantly used with propranolol. A maximum, 3‐fold, variation was observed at the 40‐mg dose level and decreased linearly with dose to an only 1.3‐fold variation at doses exceeding 600 mg/day. The fact that the oral dose of propranolol can be used to predict a very narrow plasma concentration range indicates a very uniform pattern in the way patients handle propranolol, a pattern that could prove useful as an aid in dose selection as well as a basis for an evaluation of patient compliance.

The Role of Cis-Trans Isomerization of Peptide Bonds in the Coil ⇄ Triple Helix Conversion of Collagen

Abstract: The collagen-like portion of a peptide which comprises the amino-terminal precursor-specific region of bovine type III procollagen, showed an unusually fast, concentration independent and fully reversible triple helix ⇄ coil transition. A set of three interchain disulfide bridges probably provides an effective nucleus for triple helix formation. Refolding occurred in two kinetic phases. The first one was not resolved (half time < 5 s) and the second one had a half time of about 90 s at 20 °C. When measurements were performed in phosphate buffer pH 7.0, the amplitudes of both phases were of comparable size. Activation energies for the rate constant of the slow phase ranging from 44kJ/mol at 40–30 °C to 70 kJ/mol at 15–5 °C were observed. In the presence of 4 M guanidine-HCI, essentially no fast phase was found and the activation energy was 97 ± 13 kJ/mol. The occurrence of two kinetic phases was explained by a model mechanism in which a stretch of helix between the nucleus and the first cis peptide bond is formed quickly. In the following slow phase, cis-trans isomerization at the junction of a triple helical to coiled region becomes rate-limiting. This interpretation was supported by temperature double-jump experiments. The reformation of cis peptide bonds, after a fast destruction of the triple helix, was paralleled by an increase in the amplitude of the slow phase. These findings qualitatively agree with results on the role of cis-trans isomerization in the folding of ribonuclease [Brandts, J. F., Halvorson, H. R. & Brennan, M. (1975) Biochemistry, 14, 4953–4963]. The observed curved Arrhenius plot was quantitatively explained by the normal activation energy of cis-trans isomerization (85 kJ/mol) and the contribution of the negative enthalpy of the pre-equilibria between partially helical species formed in the fast phase. Our data suggest that for long collagen molecules, the fast phase is negligible and cis-trans isomerization sets an upper limit for the rate of triple helix formation in vivo. At 37 °C the minimum time estimated for complete folding is in the range of minutes and comparable with the rate of biosynthesis. At low temperatures the slow rate of cis-trans isomerization may govern the intracellular formation of native collagen molecules to a greater extent, if no other mechanisms maintain the trans configuration of peptide bonds after release of the chains from the ribosomes.

Three Conformationally Distinct Domains in the Amino-Terminal Segment of Type III Procollagen and Its Rapid Triple Helix ⇄ Coil Transition

Abstract: Peptide Col 1–3 which comprises the whole amino-terminal precursor-specific region of bovine type III procollagen was isolated after digestion of type III procollagen with bacterial collagenase at 37 °C. Further treatment of Col 1–3 with collagenase at 55 °C released two non-collagenous segments, Col 1 and Col 2. Ultracentrifugal and circular dichroism (CD) studies indicated that Col 1–3 consists of three chain constituents associated to each other by both disulfide bridges and non-covalent bonds. The three interchain disulfide bridges are located in fragment Col 2, i.e. near the carboxyl end of the precursor-specific region. The CD spectrum indicates a random coil structure for peptide Col 2. Peptide Col 1 possesses five intrachain disulfide bridges and shows a CD spectrum indicating aperiodic structural elements and β structure. The non-covalent association of the peptide chains is due to a triple helix formed from a collagenous segment (Col 3) located between the Col 1 and Col 2 domains of the peptide. This triple helix has a melting temperature of 53 °C which decreases by about 20 °C when the peptide is reduced and S-carboxymethylated. The thermal transition of Col 1–3 is accounted for by a non-staggering zipper model without nucleation difficulty, using a standard enthalpy of triple helix formation of δHθ=− 35 kJ/mol tripeptide unit or by an all or none mechanism with δHθ=− 12.5 kJ/mol tripeptide units. Refolding of the helix from denatured Col 1–3 occurred in 100% yield with a rate constant of 8 × 10−3 S−1 at 20 °C. This is the highest rate constant yet observed for triple helix formation in vitro. Prior reduction of disulfide bonds prevented rapid helix formation.

Murine hemopoietic colonies in culture containing normoblasts, macrophages, and megakaryocytes.

Abstract: Murine marrow cells, when incubated in methylcellulose culture in the presence of erythropoietin and conditioned medium for two weeks, produced large macroscopic bursts containing normoblasts, macrophages, and often megakaryocytes. The clonal nature of these mixed colonies was supported by linearity studies and analyses of the percentages of constituent cells in different plating conditions. Time course observations and studies of the effects of L-cell-conditioned medium revealed that colony-forming cells for the mixed colonies (CFU-mix) are at earlier stages of hemopoietic development than burst-forming units (BFU-E). The mean of the modal sedimentation velocities of CFU-mix was 3.4 mm/hr and was in close agreement with that reported for the spleen colony-forming units. Almost none of the CFU-mix was in a DNA synthetic phase as measured by short-term exposure to tritiated thymidine. These results strongly indicate that CFU-mix represent a population of pluripotent hemopoietic stem cells in murine marrow.

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Abstract: The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature precrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of α-granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.

A high‐modulus polymer for porous orthopedic implants: Biomechanical compatibility of porous implants

Abstract: A high-modulus polymer, polysulfone, was evaluated as a porous bone implant material. The bone ingrowth into canine cortical pellets of sintered polysulfone particles was assessed by microradiography and histology. The shear strength of the porous polysulfone-bone interface was determined by push-out and pull-out tests of cortical and trochanteric implants, respectively. Results indicated that the bone ingrowth into porous polysulfone specimens proceeded in such a fashion as to mimic the normal repair at the site. Mechanical testing of cortical and cancellous implants revealed that the interfacial shear strength of the porous polysulfone-bone composite was similar to that achieved using porous metals.

The Rieger syndrome.

Abstract: Fourteen patients with hypodontia and the ocular features of the Rieger syndrome were examined for the presence of systemic anomalies. A periumbilical defect that consisted of failure of the periumbilical skin to involute was seen in ten of the thirteen evaluated for the defect. Three others had scars over the umbilical area and had a history of surgery for herniation. In addition, four males in one family and one male from another family had hypospadias. None of several other anomalies reported to be components of the Rieger syndrome by other authors was detected in the fourteen patients. The mode of inheritance in the familial cases studied was compatible with autosomal dominance. The results of this study indicate that the Rieger syndrome is an autosomal dominant syndrome whose cardinal features are hypodontia, goniodysgenesis, and failure of the periumbilical skin to involute properly.

Radiation therapy control of nine patients with malignant thymoma

Abstract: Malignant thymoma is a relatively rare condition and a review of the literature reveals approximately 100 reported cases. Only a small percentage of these have been treated with megavoltage radiation therapy; therefore, it is difficult to find the necessary information to establish a proper time-dose relationship for treatment. This report deals with the radiation therapy and survival data concerning nine patients treated for malignant thymoma during a ten year period at the Medical University of South Carolina. Megavoltage irradiation in the dose range of 3500-4800 rads was employed in all patients. All gross tumor was completely resected in only three patients, two had a biopsy only, and the remaining four had subtotal resections. Local tumor control has been 100% with the average follow-up being 5.5 years and a minimum of 30 months. Three patients are dead; one from intercurrent disease, one from myasthenia gravis, and one from radiation injury to the spinal cord. One patient is alive with metastatic disease controlled by chemotherapy. The technique of radiation therapy is outlined, as well as suggested treatment policy.

Observations on the development of brainstem-spinal systems in the north american opossum†

Abstract: The North American oppossum is born 12 to 13 days after conception and and is available for 90 days or more in an external pouch where it can be observed and experimentally manipulated. It is of particular interest that the hindlimbs of the newborn opossum are very immature and remain immobile for a week or more after birth. Degeneration techniques reveal that immature brainstem axons are present within the marginal zone of the lumbosacral cord before hindlimb movements begin (our state I) and material processed for formaldehyde induced fluorescence shows that some of them transport monoamines. Several lines of evidence suggest that part of the fluorescent axons arise within the nucleus locus coeruleus. At this early stage the electron microscope reveals that all brainstem-spinal axons are small (0.1--0.4 micrometer in diameter) and unmyelinated. By the time random hindlimb movements can be observed (stage II), brainstem axons, including those transporting monoamines, can be demonstrated to have grown into limited areas of the intermediate zone of the lumbosacral cord and to arise from most of the areas contributing to them in the adult animal (horseradish peroxidase technique). Such axons are still immature and it is not yet clear that they have formed synaptic terminals. Brainstem axons continue to grow into the intermediate zone of the lumbosacral cord for some time and come to occupy all of their adult territories before thoracic transection produces obvious change in hindlimb motility (beginning of stage III). It is still another 20 days or so before thoracic transection produces spinal shock comparable to that in the adult animal. The relatively mature use of the hindlimbs and the full expression of spinal shock correlate with changes in the technique and survival time needed to demonstrate degenerating brainstem axons in experimental material.

Effect of streptozotocin on erythrocyte and retinal superoxide dismutase.

Abstract: The Superoxide dismutase (SOD) activity of rat retinae and erythrocytes was decreased by 30–40% from control levels 5 days after the induction of diabetes with streptozotocin. Homogenates prepared from lungs, liver, brains, aortas, kidneys, whole eyes and lens of streptozotocin-diabetic rats showed no change in SOD activity. Treatment with insulin for three days which restored plasma glucose levels close to normal failed to affect retinal SOD activity in diabetic rats. Retinas (rat and bovine) and erythrocytes (human and rat) showed decreased SOD activity followingin vitro incubation with streptozotocin. The need for careful differentiation of drug-induced from diabetes-induced effects in studies of experimental diabetes is reemphasized.

Ultrastructural carbohydrate cytochemistry of gastric epithelium.

Abstract: On examination with ultrastructural methods for visualizing the vicinal glycols and acid groups of complex carbohydrates, the most superficial surface epithelium of the rat gastric corpus displayed biphasic mucous droplets consisting of a cortex of hexose-rich (i.e. periodate-reactive) neutral mucosubstance and an uncharacterized denser core plus monophasic droplets with the neutral mucosubstance. In many surface epithelial cells of the foveolae, the biphasic and monophasic droplets with the neutral mucosubstance intermingled in varying proportions with monophasic droplets showing uniform periodate reactivity, a variable degree of dialyzed iron-binding--demonstrative of acidic glycoconjugate, and high iron--diamine affinity--demonstrative of sulphomucin. Deep foveolar epithelium displayed only monophasic droplets, most of which contained acidic periodate-reactive complex carbohydrate. Underlying cells, designated isthmus cells, exhibited monophasic or occasional biphasic granules containing sulphated, hexose-rich mucosubstance. Nascent droplets or granules near the Golgi zone differed from the mature organelles in the distribution of the glycoconjugate. Mucous neck cells occupied a deeper stratum and displayed a uniform population of monophasic mucous droplets with a loose meshwork of neutral mucosubstance. Techniques for demonstrating hexoses ultrastructurally stained all Golgi cisternae in the mucigenic epithelium, showing increasing reactivity toward the maturing face. Distinctive cisternae with moderate reactivity in the Golgi complex of isthmus cells were interpreted as GERL. Acidic mucosubstances were visualized only in the inner, mature cisternae of the Golgi complex of cells storing acidic glycoconjugates, and not in cisternae interpretable as GERL. The apical plasmalemma of isthmus cells uniquely exhibited abundant sulphated glycoconjugate and that of parietal cells revealed a less prominent, periodic neutral mucosubstance. Lateral and basal plasmalemmae varied from unstained to slightly reactive; basement membranes showed moderate reactivity with methods for visualizing complex carbohydrates. Abundance of glycogen further characterized surface epithelial cells of the corpus and of some parietal cells.

Intravascular papillary endothelial hyperplasia: light and electron microscopic observations of a case.

Abstract: The ultrastructure of a case of intravascular papillary endothelial hyperplasia is presented. The papillae and septa within the lesion showed a similar organization. The surface was lined by a nonstratified layer of plump endothelial cells. Immediately beneath lay cells and processes of pericytes rich in thin filaments with fusiform densities, resembling smooth muscle cells. Primitive mesenchymal cells were observed to lie among the pericytes. The stroma beyond the subendothelial layer was collagenous and contained fibroblasts. The ultrastructure of the cells and their organization closely resembles human granulation tissue.

Effects of naloxone on ethanol induced alterations of locomotor activity in C57BL/6 mice.

Abstract: Ethanol (2 g or 3 g/kg) or water vehicle was injected intraperitoneally into C57BL/6 mice 15 min after injections of naloxone, a narcotic analgesic antagonist, or its saline vehicle. Locomotor activity was monitored for 60 min beginning 30 min (Experiment 1) or immediately (Experiment 2) following the ethanol injection. In both experiments, animals injected with the lower dose of ethanol were more active than controls during the second half of the activity test. Animals injected with the high dose of ethanol were less active than controls during the first half of the activity test but returned to control levels or above during the second half of the test. Naloxone at the doses used injected 45 min prior to the activity test (Experiment 1) did not alter locomotor activity and did not influence ethanol induced activity changes. When injected 15 min prior to testing (Experiment 2), however, naloxone alone produced a transient reduction in activity observed only during the first half of testing. During the second half of testing all animals injected with naloxone had activity levels similar to controls and lower than those of animals injected with ethanol in the absence of naloxone. Hence, it appears that naloxone at a dose and time period which does not alter the locomotor activity of mice is capable of blocking ethanol induced excitatory effects.

Relaxin-dependent adenosine 6',5'-monophosphate concentration changes in the mouse pubic symphysis.

Abstract: The amount of cAMP in the pubic symphyses of estrogen-primed mice increases after injection of the hormone relaxin. This relaxin-induced increase in symphyseal cAMP was observed in immature, mature, and ovariectomized mature mice and could be prevented by prior ip injection of rabbit antibodies to porcine relaxin or by treatment of relaxin with dithiothreitol. The highest level of cAMP was measured 30 min after relaxin injection; the level of measurable cMAP then diminished rapidly. Estrogen-priming of the mice was not a prerequisite for a relaxin-induced response to occur. Relaxin administration did not increase the level of cAMP in a non-target tissue such as liver, nor could an increase in cAMP in the pubic symphysis be elicited by injection of insulin, a protein of similar size and structure, or glucagon, a known stimulator of liver cAMP levels.

Relationship between abrasive wear and microstructure of composite resins.

Abstract: The in vitro abrasion resistance of seven commercial composite resin restorative materials has been measured. Analysis of the composite microstructures shows that abrasion rates are dependent upon the size, hardness, and volume fraction of particles in the material. The most abrasion-resistant composites contain a high volume fraction of large, hard particles.

Combined Tricyclic—MAOI Therapy for Refractory Depression: A Review, with Guidelines for Appropriate Usage

Abstract: February-March, 1978 143 I T is known that a considerable number of depressed patients prove refractory to monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and electroconvulsive therapy or cannot achieve sustained benefit from the individual use of these modalities. This group is said to range from 10 to 28 per cent of patients so treated.12 The use of TOA-MAOI combination in these patients raises some important theoretical considerations. First of all, the efficacy of combined therapy has yet to be tested by double-blind controlled studies (although two such studies are reportedly under way). Several authors, however, have found combination therapy to be highly beneficial in previously refractory patients, using the patients themselves as controls.31#{176} The issue concerning the intrinsic safety and value1’ of MAOIs is beyond the scope of this paper, but it may be safe to say that its use in combination therapy with TCAs does not outweigh the

A model of high affinity glutamic acid transport by cortical synaptosomes from the Long-Evans rat.

Abstract: — The initial velocity of uptake of L-glutamic acid by cortical synaptosomes from the Long-Evans rat has been measured as a function of sodium and glutamic acid concentration. These data were then fitted to the rate equation from each of several possible models, and the model giving minimum error identified. The major predictions from the best fit model are as follows: (1) The order of combination with the carrier should be sodium, sodium, glutamic acid. (2) Uptake should be 100% dependent on the presence of sodium in the incubation medium. (3) At a finite concentration of sodium, Vmx should be independent of the sodium concentration, which implies that translocation of the carrier across the membrane is independent of the sodium concentration. (4) Lineweaver-Burk plots should be linear with slopes depending on the sodium Concentration. (5) There should be co-transport of two sodium ions with each glutamic acid molecule. (6) The dependence of Kt, on the sodium concentration should have the following form: Kt= A/[Na]2+ 8/[Na] +C, where A, B, and C are constants. The results differ substantially from those previously reported for Sprague-Dawley rats. Kt, is 2–5 times less than that for Sprfague-Dawley rats, and the relation of sodium to Kt, is basically different. We also find a coupling ratio of two, whereas previous studies found a coupling ratio of one. Thus the results raise the possibility that there are fundamental differences between Sprague-Dawley and Long-Evans rats with regard to the mechanism by which sodium participates in amino acid transport.

Some problems inherent in transport studies in synaptosomes

Abstract: — A technique utilizing a 30-place manifold has been developed to study synaptosomal transport; some problems associated with such studies have been identified and clarified. The time course of L-glutamic acid uptake has been used to test variations in experimental protocol. Synaptosomes apparently become increasingly labile with increased time of incubation. This is indicated by a drop in the curve of uptake vs time after 8–12 min. Ninety seven to 98% of the glutamate taken up from a 10−6m solution is released by osmotic shock. Synaptosomes can be stored in 0.32 m ice-cold sucrose suspension for periods up to 50 min without decline in measured uptake. Storage for 3 h or more results in a very substantial decline in measured uptake. Neither the decline in measured uptake with time, nor the decline with storage, is prevented by increasing the osmolarity of the solutions used or by use of synaptosomes from the initial 1085 g supemate rather than after sedimentation and resuspension. Although prewarming synaptosomes at 30°C for 20 min prior to their use lessened or eliminated the decline following peak uptake, the difference between stored and non-stored synaptosomes was not improved. Uptake was also much less when synaptosomes were used from the first supernate or when warmed prior to their use. Storage of tissue prior to homogenization resulted in synaptosomes that gave minimal reductions in measured uptake. Washing synaptosomes after separation from incubation medium resulted in a variable loss of substrate radioactivity, depending on such variables as brand of filter, pore size, composition of wash solution, and temperature of wash solution. The results support the hypothesis that washing causes lysis of a portion of the synaptosomes. However, with Millipore filters (0.45 μm) and a 30°C Krebs-Henseleit wash solution, the loss caused by washing is minimized (about 15%). Measured uptake is found to depend on the type of filter used. Uptake is much greater with Millipore 0.45 pm filters than with Gelman 0.45 μm filters. Use of Nuclepore (0.4 μm) filters results in measured uptakes only about 5% of that when Millipore 0.45 μm filters are used. With Millipore filters, 0.30 μm pore size filters gave uptakes only 68% of that using 0.45 pm pore size filters.