Showing papers by "University of Maryland, Baltimore published in 1980"

Metabolism of Proline and the Hydroxyprolines

Abstract: PERSPECTIVES AND SUMMARY 1006 INTRODUCTION 1007 PROLINE 1008 Proline Biosynthesis: The Glutamate Pathway 1008 Proline Biosynthesis: The Om Pathway 1015 Proline Degradation: The P5C Pathway 1018 Proline Degradation: Reguiation 1020 Proline Degradation: Other Pathways 1023 4-HYDROXYPROLlNE 1024 Natural Occurrence 1024 Metabolism General Features 1025 Catabolism of Free 4-Hyp 1034 3-HYDROXYPROLINE 1041 Metabolism 1042 3,4-DIHYDROXYPROLINE 1044

Some properties of potassium-stimulated calcium influx in presynaptic nerve endings.

Abstract: Potassium-stimulated 45Ca entry into rat brain synaptosomes was measured at times ranging from 1 to 60 s. The K-rich solutions were used to depolarize the synaptosomes. Backflux of 45Ca from the synaptosomes was negligible during the first 10-20 s of incubation. An initial ("fast") phase of K-stimulated Ca entry, lasting from 1 to 2 s was observed. This phase was inhibited by low concentrations of La (KI approximately equal to 0.3 microM). It was also abolished ("inactivated") by incubating the synaptosomes in depolarizing solutions (containing veratridine, gramicidin, or elevated [K]o) before the addition of 45Ca. An additional long lasting ("slow") phase of K-stimulated Ca entry was also detected. This "slow" Ca entry was much less sensitive to La (KI > 100 microM) and was not affected by depolarizing the synaptosomes before the addition of 45Ca. The rate of influx during the fast phase was about four times the rate of Ca influx during the slow phase. Neither the fast nor slow phase of Ca entry was sensitive to tetrodotoxin (10 microM), a potent blocker of Na channels, but both phases were inhibited by Ni, Mn, Mg, and other agents that block Ca channels. The data are consistent with the presence of two distinct populations of voltage-regulated, divalent cation-selective pathways for Ca entry in presynaptic brain nerve endings.

Platelet transfusion therapy. One-hour posttransfusion increments are valuable in predicting the need for HLA-matched preparations.

Abstract: Seventy-nine platelet transfusions to 73 thrombocytopenic patients with cancer were analyzed to determine whether a platelet count obtained one hour after transfusion could help differentiate between alloimmunization and other clinical factors that result in rapid platelet destruction. These transfusions were selected because 18- to 24-hour increments were inadequate in response to fresh, random donor platelets. A corrected count increment (CI) (CI=[posttransfusion count—pretransfusion count]×body surface area [sq m]/platelets transfused×10 11 ) at one hour of 10×10 3 /μL or greater was associated with absence of lymphocytotoxic antibody, whereas increments of less than 10×10 3 /μL were generally associated with high levels of strongly cytotoxic antibody. HLA-matched transfusions produced no improvement in increments when the previous one-hour CI had been 10×10 3 /μL or greater, whereas in the other group significantly better increments were obtained. A one-hour posttransfusion count is a simple test that correlates well with the presence of antibody against HLA antigens, is valuable in predicting the need for HLA-matched platelets, and helps avoid wasteful, empirical use of such transfusions. ( JAMA 243:435-438, 1980)

Cold face test in the assessment of trigeminal‐brainstem‐ vagal function in humans

Abstract: Study of the reflex heart rate response in humans to apneic facial immersion (simulated diving) and its modifications showed that bradycardia caused by simple application of cold compresses to the face (cold face test) correlated well with that produced by the simulated diving reflex. Bilateral application of cold stimulus to the individual divisions of the trigeminal nerve revealed the ophthalmic division to be the most sensitive pathway for this reflex. The cold face test was standardized in 50 normal individuals and further validated in 10 patients by comparison with the simulated diving reflex, the Valsalva maneuver, and administration of atropine. Patients with diabetes mellitus, brainstem stroke, multiple sclerosis, or Shy-Drager syndrome developed less than normal bradycardia or minimal tachycardia in response to the cold facial stimulus. The cold face test is a novel, simple, safe, and economical test of the integrity of trigeminal-brainstem-vagal reflex pathways, can be utilized practically to assess vagal and brainstem dysfunctions, and has the special advantage of being applicable even in an uncooperative or comatose patient.

Anatoxin-a: a novel, potent agonist at the nicotinic receptor.

Abstract: Anatoxin-a, a semirigid, bicyclic amine, caused a depolarizing blockade of the indirectly elicited twitch in frog sartorius muscle. Concentration-response studies of contracture in the rectus abdominis and depolarization in the sartorius muscles of the frog showed that it is the most potent of the nicotinic agonists. It produced desensitization, and the kinetic and steady-state parameters found from chronically denervated rat soleus muscle were similar for anatoxin-a and acetylcholine. When endplate regions of frog sartorius fibers were voltage clamped, anatoxin-a induced endplate currents and concomitant increases in endplate current noise. Fourier analysis of this noise revealed that the average single channel lifetime was indistinguishable from that induced by acetylcholine; the single channel conductance was about 25% less for the toxin compared to acetylcholine. Analysis of nerve evoked endplate currents and spontaneous, miniature endplate currents indicated that the toxin did not alter the time constants for decay or the linearity of the current-voltage relationship. We conclude from this evidence that anatoxin-a does not block the open ion channel and does not produce a voltage-dependent blockade of the closed ion channel. The activity of this unusual agonist may be related to the coulombic and hydrogen bond free energies of binding to the receptor.

The Effect of Particle Size on the Osteogenic Activity of Composite Grafts of Allogeneic Freeze-Dried Bone and Autogenous Marrow,

Abstract: This study was carried out to determine if particle size is a factor to be considered in the evaluation of the osteogenic activity of freeze-dried bone allografts (FDBA) and, if so, whether small particles enhance or inhibit osteogenesis. Small particle FDBA (100-300 microns) plus marrow and large particle FDBA (1000-2000 microns) plus marrow were placed in plexiglass diffusion chambers secured to the femurs of six Rhesus monkeys. Control chambers contained either marrow alone or were left empty. Two animals were given injections of oxytetracycline hydrochloride at 5 and 7 weeks to obtain intravital osseous labeling. All chambers were removed after 8 weeks. Ten chambers were evaluated for new bone formation by fluorescent microscopy. The contents of 15 additional chambers were evaluated by single blind technique for presence or absence of bone resorption and ossification. The results indicated that there was significantly more new bone formation associated with small particle FDBA (100-300 microns) plus autogenous marrow than with large particle FDBA (1000-2000 microns) plus autogenous marrow. In addition, small particle FDBA (100-300 microns) plus autogenous marrow tended to display more resorption than large particle FDBA (1000-2000 microns) plus autogenous marrow. It was concluded that within the parameters of this study, small particles of FDBA enhance osteogenesis. This study also demonstrated that particle size is a variable to be considered when comparing the osteogenic potential of freeze-dried bone allografts.

Effect of ovariectomy on plasma LH, FSH, estradiol, and progesterone and medial basal hypothalamic LHRH concentrations in old and young rats.

Abstract: Resting plasma concentrations of LH, FSH, estradiol, and progesterone and medial basal hypothalamic concentrations of LHRH (MBH-LHRH) were measured by RIA in 8- to 12-month-old female rats which had b

Lactate: A major product of glutamine metabolism by human diploid fibroblasts

Abstract: Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.

Cast Metal Resin-Bonded Retainers for Posterior Teeth

Abstract: The design, fabrication, and bonding of the cast resin-bonded retainer for posterior teeth are described, and the advantages of this development in restorative dentistry are discussed.

Binding sites for thyrotropin-releasing hormone in sheep nucleus accumbens resemble pituitary receptors.

Abstract: TRH binds to sites in the nucleus accumbens-septal area of sheep brain. These sites appear to represent receptors for at least some of its behavioral effects in other species and are very similar to sheep pituitary receptors. All measurements were performed on ice to prevent peptide degradation. High affinity [3H]TRH binding in brain regions was distinguished from interfering low affinity binding by use of [3-Me-His2]TRH, a more potent and specific analog, in blank tubes at a 1-microM concentration. The nucleus accumbens-septal area, particularly the nucleus accumbens itself, showed the highest binding of any of a variety of brain regions surveyed. Binding sites in both nucleus accumbens and anterior pituitary had an equilibrium dissociation constant of about 20-40 nM, a rate constant for association of about 1-3 x 10(6) M-1 min-1, and a rate constant for dissociation of about 0.07 min-1. Seventeen TRH analogs showed closely similar potencies in competing for binding in the two tissues. Six weak analogs appeared to be more potent in the nucleus accumbens than in the pituitary, but this was an artifact of their relatively greater potency in competing for low affinity binding sites which are absent in pituitary. The only major difference between the high affinity binding sites in the two tissues was in their concentration, which was about 2- to 3-fold higher in the pituitary.

Metabolism of benzo[a]pyrene by cultured tracheobronchial tissues from mice, rats, hamsters, bovines and humans.

Abstract: The metabolism of benzo[a]pyrene (BP)4by cultured tracheobronchial tissues from different species - human, bovine, hamster, rat and mouse - has been investigated. The total metabolism, as measured by both organic solvent-extractable and water-soluble metabolites of BP, was substantial in the respiratory tract from humans and from animal species susceptible to the carcinogenic action of BP. The ratio of organic-extractable metabolites to water-soluble metabolites was greater than one in hamster, human and C57BI/6N mouse, but less than one in rat, bovine and DBA/2N mouse, suggesting that determination of both activation and deactivation pathways are important in assessing carcinogenic risk of a chemical. Sulphate esters and glutathione conjugates were the major water-soluble metabolites in all animal species; tetrols and diols were the major organic extractable metabolites. The level of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, the proximate carcinogenic form of BP, was three times higher in C57BI/6N than in DBA/2N mouse trachea. Trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major metabolite formed by cultured hamster trachea. The binding levels of BP to cellular DNA were quite similar in all tissues, although slightly higher binding was observed in hamster trachea. Wide inter-individual variation in the binding of BP to DNA was seen in tissues from outbred species. The major BP-DNA adducts in all animal species were formed by interaction of benzo[a]pyrene diol-epoxide with the 2-amino group of deoxyguanosine. Both stereoisomeric forms of (±)-(7β,8α)-dihydroxy-(9α, 10α)-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE I) reacted with deoxyguanosine, the (7R)-form being the most reactive. No difference in the relative distribution of the various adducts was seen between the species except in the CD rat, where BPDE-deoxyadenosine adducts accounted for 20% of the total modification. In cultured hamster trachea the persistence of the different adducts was similar. In conclusion, the metabolism of BP is qualitatively similar in tracheobronchial tissues from both humans and animal species in which BP has been experimentally shown to be carcinogenic.

Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts-1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

Abstract: Two attenuated influenza A donor viruses, the A/Udorn/72 ts-1A2 and the A/Ann Arbor/6/60 cold-adapted (ca) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts-1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID(50)) for each recombinant was approximately 10(5.0) 50% tissue culture infective doses. The virus shed by the ts-1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts-1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID(50) and approximately 70% of ca or ts vaccinees who received 10 to 32 HID(50) of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.

Levels of batrachotoxin and lack of sensitivity to its action in poison-dart frogs (Phyllobates).

Abstract: Batrachotoxin is present in remarkably high amounts in the skin of Phyllobates terribilis. Levels of batrachotoxin tend to be reduced when P. terribilis is maintained in captivity, but even after being confined for up to 6 years, these frogs were still at least five times more toxic than other Phyllobates species used by natives for poisoning blowgun darts. Batrachotoxin was not detectable in F1 progeny reared to maturity in captivity. Nerve and muscle preparations from wild-caught frogs and from the nontoxic F1 frogs were both insensitive to batrachotoxin. The regulatory site controlling sodium-channel activation and permeability appears to have been minimally altered to prevent interaction with batrachotoxin, but is still sensitive to other sodium conductance activators (veratridine, grayanotoxin) to which the frogs arenot exposed naturally.

Sites of action of phencyclidine. II. Interaction with the ionic channel of the nicotinic receptor.

Abstract: The effects of phencyclidine (PCP) were studied on the endplate current (EPC), miniature endplate current (MEPC), and synaptic noise of the frog sartorius muscle and on the binding of [3H]perhydrohistrionicotoxin ([3H]H12-HTX) to the ionic channel of the acetylcholine (ACh) receptor in electric organ membranes of Torpedo ocellata. PCP decreased the peak amplitude of the EPC in a voltage- and time-dependent manner, caused nonlinearity in the current-voltage relationship, accelerated the decay time constants of the EPC and MEPC, and shortened the mean lifetime of the single ionic channel. PCP also inhibited binding of [3H]H12-HTX to the ionic channel of the ACh receptor with a Ki, of 6.9 µM. When carbamyicholine was present to activate the ACh receptors, the Kd value for PCP binding to ionic channel sites was reduced from 10.3 ± 4.2 to 2.0 ± 1.3 µM, thus showing higher affinity for the activated ionic channel sites. In addition, PCP also reacted with the closed ionic channel since a time-dependent effect on EPC amplitude in hyperpolarized membranes was observed even before the ACh receptor was activated. Further, PCP depressed peak EPC amplitude more markedly than it shortened the EPC decay time constant, thus disclosing that the depression of the former cannot be accounted for totally by the action of the agent on the open conformation of the ionic channel. A hybrid model was proposed to account for the interactions of PCP with the open and closed states of the ionic channel of the ACh receptor. The actions of PCP on both states of the ionic channel are qualitatively similar to those seen with histrionicotoxin.

Interferon treatment inhibits glycosylation of a viral protein

Abstract: Earlier, we reported a 30-200-fold reduction in the yield of infectious vesicular stomatitis virus (VSV) released from L cells treated with 10-30 reference units ml-1 of interferon (IFN); however, in these cultures virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein and viral transcriptase, was inhibited less than 10-fold. There was biochemical and morphological evidence of a significant reduction in glycoprotein (G) and membrane protein (M) of VSV particles released from IFN-treated cells. We compare here the effects of tunicamycin (TM) and IFN in L cells. Treatment with TM or IFN reduced the production of infectious VSV particles, decreased the amount of G and M proteins in VSV released from treated cells, and inhibited an early step in the formation of asparagine-linked oligosaccharide chains, the incorporation by membrane preparations from treated cells of N-acetylglucosamine into glycolipids with the properties of dolichol derivatives.

Hemagglutination and colonization factors in enterotoxigenic and enteropathogenic Escherichia coli that cause diarrhea.

Abstract: Controversy exists as to whether all strains of enterotoxigenic Escherichia coli must possess colonization factor antigen pili I or II (CFA/I, CFA/II) in order to be virulent for humans. Failure to detect CFA/I or CFA/II in enterotoxigenic strains from human diarrhea has been explained by some as due to plasmid loss and by others as evidence that CFA/I and CFA/II pili are not prerequisites for human virulence in all enterotoxigenic strains of E. coli. Seven enterotoxigenic and three enteropathogenic strains of E. coli that have been used in volunteer challenge studies were tested for CFA/I, CFA/II, and type 1 somatic pili after culture on solid agar and in broth. Six of the seven enterotoxigenic and two of the three enteropathogenic strains caused diarrhea in humans. Of these eight virulent strains, one produced CFA/I, and one had CFA/II. Threre remained four enterotoxigenic and two enteropathogenic strains that unequivocally caused diarrhea while lacking CFA/I or CFA/II. In such strains other organelles or surface properties must be operative to promote adhesion to and colonization of small intestinal mucosa. CFA/I and CFA/II are not prerequisites of virulence for all E. coli strains that cause diarrhea in humans.

Progesterone and Estrogen Secretion by Cultured Monkey Ovarian Cell Types: Influences of Follicular Size, Serum Luteinizing Hormone Levels, and Follicular Fluid Estrogen Levels

Abstract: Rhesus monkeys were laparotomized before and during the LH surge, and the thecal layer, stroma, and granulosa cells were removed and cultured for 8 days, separately or together, in the presence or absence of 5 × 10-7 M testosterone. In each experiment (n = 30), estrogen and progesterone were measured in the follicular fluid and serum immediately before follicular harvest. The oocyte was harvested from the largest (presumed preovulatory) follicle present; its degree of maturity was determined using a stereomicroscope, followed by culture of the oocyte for 2 days. Follicles containing granulosa cells which failed to attach and grow, or degenerated oocytes (at the time of harvest or degenerated in culture) were not used and were designated atretic. After initiation of the LH surge, there was a significant rise in follicular fluid progesterone and estrogen levels, with estrogen levels reaching a peak at the time of the LH surge. One day after the LH surge, follicular fluid and serum estrogen levels dropped sh...

A developmental change in dolichyl phosphate mannose synthase activity in pig brain.

Abstract: The initial rate of dolichyl phosphate mannose biosynthesis was measured in white-matter membranes from pig brain at various ages from before birth throughout the period of most rapid brain development. Dolichyl phosphate mannose synthase activity increased from prenatal values to a maximum in 3 week-old animals, and gradually decreased to adult values after 8 weeks of age. The nature of the developmental change was investigated by enzymic and biochemical comparisons of the membrane preparations from the most active age (3 weeks) and adult controls. The specific activity of dolichyl phosphate mannose synthase in preparations from actively myelinating animals was approx. 3-fold higher than adults when mannolipid formation was assayed with saturating concentrations of GDP-[(14)C]mannose and utilizing only endogenous acceptor lipid. No major variations were found in the apparent K(m) values for GDP-mannose or exogenous dolichyl monophosphate. However, the ratio of dolichyl phosphate mannose synthase activity for myelinating animals/adult animals decreased significantly when large amounts of exogenous dolichyl monophosphate were added to the incubation mixtures. Dolichyl phosphate mannose synthase activity was also compared in white-matter membranes depleted of endogenous dolichyl monophosphate by enzymic mannosylation or treatment with butanol. When these preparations were assayed with identical amounts of exogenous dolichyl monophosphate, the dolichyl monophosphate-depleted membranes from actively myelinating animals contained only 20-30% more dolichyl phosphate mannose synthase activity. Overall, these studies strongly suggest that the developmental change in dolichyl phosphate mannose synthase activity is due primarily to the presence of a relatively lower amount of endogenous dolichyl monophosphate being accessible to the mannosyltransferase in the white-matter membranes from adult animals.

Sites of action of phencyclidine. III. Interactions with muscarinic receptors.

Abstract: The effects of phencyclidine (PCP) and phencyclidine methiodide (PCP-MeI) on contractions of longitudinal muscle of guinea pig ileum and specific binding of [3H](DL)3-quinuclidinyl benzilate ([3H]QNB) to muscarinic receptors in these muscles and rat brain were studied. PCP inhibited competitively muscarinic agonist-induced contractions of the longitudinal muscle of guinea pig ileum at concentrations below 10 µM (Ki = 0.45 µM). However, at 50 µM noncompetitive blockade of contraction was seen. In contrast, PCP-MeI inhibited ileum contractions competitively at concentrations of up to 100 µM (Ki = 0.2 µM). Both drugs inhibited binding of [3H]QNB to muscarinic acetylcholine (ACh) receptors of rat cerebral cortex and brain stem and to ileal longitudinal muscle (apparent Ki’s of from 0.8 to 3.7 µM). There was no evidence of noncompetitive receptor binding inhibition at PCP concentrations which produced noncompetitive inhibition of smooth muscle contraction. There were no indications of multiple receptor populations, cooperative interactions, or receptor isomerization with either drug. PCP-MeI was slightly more potent in its competitive muscarinic actions than PCP. Although the dissociation constant (Kd) of PCP from muscarinic receptors in brain cortex was significantly lower than in brain stem, the Kd values for PCP-MeI were the same. Treatment with N-ethylmaleimide did not affect the inhibition of [3H]QNB binding to muscarinic receptors by PCP. Based on several characteristics of the inhibition by PCP and PCP-MeI of [3H]QNB binding to rat brain muscarinic receptors and their inhibition of the contractions of longitudinal muscle of guinea pig ileum, PCP and PCP-MeI are considered antagonists of muscarmnic receptors in rat brain.