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Biomedical subjects

A E Johnson

Publications and source records attributed to A E Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1989

Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel.

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.

Cross-Linking Reagents↗
PubMed1989

Estradiol modulation of oxytocin binding in the ventromedial hypothalamic nucleus of male and female rats.

It has been suggested that estradiol and oxytocin (OT) may interact as neuroendocrine components in the regulation of sexual behavior. In the present study the effect of estradiol benzoate (EB) treatment (50 micrograms/kg body weight/2 days) on [3H]-OT binding was evaluated in adult and 21-day-old gonadectomized male and female rat brains. Coronal sections through the ventro-medial nucleus of the hypothalamus (VMN) were analyzed in three different section planes. EB priming induced an increase in [3H]-OT binding in the VMN of both male and female rats. Greater binding site density and significant EB effects were found in the most caudal plane where the ventrolateral portion of the VMN is well defined at both ages. OT binding in the central amygdaloid nucleus was not affected by this treatment but higher binding levels were found in the most caudal sections irrespective of hormonal status or sex. No sex differences were detected in OT binding in the VMN of basal or EB-treated animals. These results suggest that a dose of EB which activates female sexual behavior in female but not in male rats is able to induce similar levels of OT binding in the VMN of animals of both sexes.

Aging↗
PubMed1989

Testosterone modulates oxytocin binding in the hypothalamus of castrated male rats.

Oxytocin (OT) binding sites are modulated by estrogens in several brain regions including the ventromedial hypothalamic nucleus (VMN) in both male and female rats. To further study steroid regulation of OT receptor binding, we examined the effect of androgen replacement in castrated male rats on OT binding with quantitative autoradiographic methods. Castrated adult male rats were treated with either 250 micrograms testosterone propionate (TP) or oil for 2 days and killed 48 h after the last injection. Brain sections through the preoptic area and VMN were labeled with 5.0 nM[3H]-OT +/- 5.0 microM unlabeled OT or 1.0 microM[Thr4,Gly7]OT and apposed to tritium-sensitive film for 7 weeks. Results of this study show that TP increased [3H]-OT binding up to 5-fold in the ventrolateral VMN and 4-fold in the bed nucleus of the stria terminalis. In addition [Thr4,Gly7]OT completely displaced [3H]-OT binding in the VMN indicating that binding in this brain region was specific to OT receptors. Because estrogens also increase OT receptor binding in male rats, it is possible that TP affects OT binding after being converted by aromatase to estradiol.

Animals↗
PubMed1989

Anatomical localization of the effects of 17 beta-estradiol on oxytocin receptor binding in the ventromedial hypothalamic nucleus.

Oxytocin (OT) neurotransmission plays a role in the facilitation of steroid-dependent sexual receptivity in the rat. One way in which the ovarian steroid 17 beta-estradiol (E2) has been shown to modulate OT transmission is by increasing OT receptor binding in certain brain areas involved in the regulation of female sexual behavior such as the ventromedial hypothalamic nucleus (VMN). This study was undertaken to describe the distribution of OT receptors within the VMN that are regulated by physiological levels of E2. With quantitative autoradiographic methods, we measured [3H]OT binding in ovariectomized female rats implanted with Silastic capsules containing cholesterol, 5% E2, or 100% E2. In addition, plasma E2 levels, pituitary progestin receptor binding, and uterine weights were measured in animals from each treatment group. Results of this study showed that physiological levels of E2 increased [3H]OT binding in caudal regions of the ventrolateral VMN and stimulated maximal uterine growth and pituitary progestin receptor binding. However, in more rostral VMN sections, E2 induced a dose-dependent increase in [3H]OT binding. These data suggest that ovarian steroids sensitize the brain to OT by increasing OT receptor binding in certain brain areas involved in the regulation of sexual receptivity.

Animals↗
PubMed1989

Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat.

Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay of OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.

Animals↗
PubMed1989

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study.

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.

Adult↗
PubMed1988

Alpha 1- and alpha 2-noradrenergic receptors in steroid-sensitive brain areas: development and response to estradiol-17 beta benzoate in neonatal guinea pigs.

Immature female guinea pigs are relatively insensitive to the effects of ovarian steroids on certain reproductive processes including the facilitation of sexual receptivity and the induction of hypothalamic progestin receptors by estrogen. In adult guinea pigs, alpha-receptor-mediated noradrenergic transmission is known to affect both of these processes. In the experiments reported here, we investigated the possibility that age-related differences in alpha 1- and alpha 2-noradrenergic receptors might underlie the insensitivity of neonates to ovarian steroids. With tritium-sensitive film autoradiography, the distribution of alpha 1- and alpha 2-receptors in neonatal and adult female guinea pigs was compared and the effects of exogenous estradiol-17 beta on alpha-receptor binding was examined in neonatal guinea pigs. The results of these experiments showed that in animals not treated with estrogen the binding of both alpha-receptor subtypes differed between adults and neonates in several brain regions including areas known to be involved in the regulation of reproduction. In all regions where differences occurred, alpha-receptor levels were higher in neonatal females than in adult females. In addition, in contrast to previously reported results in adults, estrogen did not affect alpha-receptor binding in any region of neonatal guinea pig brain.

Animals↗
PubMed1988

Alpha 1- and alpha 2-noradrenergic receptor binding in guinea pig brain: sex differences and effects of ovarian steroids.

Noradrenergic transmission mediated by alpha 1-receptors is involved in the regulation of ovarian steroid-dependent reproductive processes in female guinea pigs. In addition, estradiol treatment is known to affect alpha 2-receptor binding in some steroid-sensitive regions of guinea pig brain. In these experiments, we further assessed the effects of ovarian hormones on alpha-noradrenergic receptors with quantitative autoradiographic methods. Binding of alpha 1- and alpha 2-noradrenergic receptors was measured in male and female guinea pigs treated with either estradiol or oil to determine if sex differences in steroid sensitivity might be related to differences in the distribution or modulation of alpha-receptors. In a second experiment, the possibility was tested that progesterone might alter the sensitivity to norepinephrine by modulating alpha 1- or alpha 2-receptor binding in estradiol-primed female guinea pigs. Results of these studies showed that estradiol treatment reduced alpha 1- and alpha 2-receptor binding of the ventromedial hypothalamic nucleus only in females and increased alpha 2-receptor binding in several preoptic nuclei of both sexes. Progesterone administration, however, had no effect on either alpha-receptor subtype. These studies indicate that estradiol-induced changes in alpha-receptor binding might be involved in steroid-dependent reproductive processes and in the differential responsiveness of males and females to ovarian hormones. Changes in these receptors do not appear to be involved in the stimulatory effects of progesterone on neuroendocrine processes.

Animals↗
PubMed1988

Delayed manifestation of Senecio-induced pyrrolizidine alkaloidosis in cattle: case reports.

Selected case reports from closely controlled experimental feedings of Senecio jacobaea (tansy ragwort), S longilobus (threadleaf groundsel) and S riddellii (Riddell's groundsel) to cattle are presented to show that all 3 of these pyrrolizidine alkaloid-containing plants may not necessarily induce proximate toxicity, but may cause typical signs and death many months after the plants are ingested. Results of the experimental intoxications indicate that if the daily dosage is of sufficient size to insult hepatocytes, a chronic lethal dose of plant may be eaten in only a few days. Early signs of intoxication may be absent, or mild and transitory, and serum enzyme changes that reflect liver injury may not always be detectable. Latent pyrrolizidine alkaloidosis signs can develop many months after the plants are eaten, and death imminent soon thereafter. The prepatent period and eventual time of death appears to be dependent on the time-dose relationship that determines the rate of progressive hepatocellular injury.

Animals↗
PubMed1987

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex.

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.

Animals↗
PubMed1987

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents.

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adamantane↗
PubMed1987

Noradrenergic regulation of alpha 1-receptors during the postnatal development of the guinea pig.

In guinea pig brain, alpha 1-noradrenergic receptor concentrations undergo region-specific fluctuations during the first weeks of postnatal life. However, the factors involved in the regulation of these receptors have yet to be identified. In this study, the ontogeny of one possible regulatory factor, norepinephrine, was examined in relation to postnatal changes in alpha 1-receptor levels in several different regions of guinea pig brain. Results from these studies showed that while the activity of the noradrenergic system increased throughout the first weeks of postnatal development in each brain area examined, the concentration of alpha 1-receptors decreased in preoptic area and hypothalamus and increased in cortex. In subsequent experiments, the effects of noradrenergic lesions with 6-hydroxydopamine on alpha 1-receptor levels were assessed to examine the possibility that alpha 1-receptors are differentially sensitive to noradrenergic stimulation in cortex and preoptic area/hypothalamus in immature guinea pigs. Noradrenergic lesions which reduced norepinephrine levels by 87-94% resulted in significant elevations in alpha 1-receptors in all regions examined. These results are discussed with reference to the anatomical distribution of alpha 1-receptors and their regulation by norepinephrine.

Animals↗
PubMed1986

A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle.

Photoreactive moieties were incorporated into nascent polypeptides in a wheat germ protein-synthesizing system by using a plasmid-derived preprolactin mRNA and a Lys-tRNA analog, N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA (epsilon ANB-Lys-tRNA). The presence of the abnormally large amino acid side chains in the nascent chains did not impair function: complete preprolactin chains were synthesized in the absence of the signal recognition particle (SRP), elongation was arrested in the presence of SRP, and SRP-dependent translocation across the membrane of the endoplasmic reticulum and signal peptidase cleavage were observed in the presence of salt-extracted microsomes. Photolysis of elongation-arrested ribosomes resulted in several light- and epsilon ANB-Lys-tRNA-dependent crosslinks. By using antibodies specific for each of the proteins, one covalent complex was shown to be a photocrosslink between the preprolactin nascent chain and the 54-kDa protein subunit of SRP. This demonstrates that the N-terminal end of a secretory protein is located adjacent to the SRP in elongation-arrested ribosomes and strongly suggests that the signal sequence is recognized by and binds to the 54-kDa subunit of SRP. The other photocrosslinks involve as-yet-unidentified proteins in the large ribosomal subunit, indicating that this method of incorporating probes provides a powerful approach to examining the environment and interactions of the nascent chain during translation and translocation across the membrane of the endoplasmic reticulum. The Lys-tRNA analog also successfully photoaffinity-labeled the Escherichia coli elongation factor Tu (EF-Tu) in the epsilon ANB-Lys-tRNA.EF-Tu.GTP ternary complex.

Affinity Labels↗
PubMed1986

A risk analysis of hexachlorobenzene-related reproductive outcomes.

Hexachlorobenzene (HCB) has been shown to produce reproductive effects in man and animals. The risks associated with hexachlorobenzene exposure have been determined for a litter using data from a feeding study by Kitchin et al. (1982). The total amount and the concentration of HCB in the litter has been estimated from a pharmacokinetic model. This estimate is consistent with the works of Kitchin et al. (1982), Courtney and Andrews (1985), and others which demonstrate that a large amount of HCB is transferred to the litter, and this quantity increases as a function of the number of lactating days. The transfer of large quantities of HCB caused a significant increase in the number of litters with greater than or equal to 10% mortality when total HCB in the litter was 29-57 mg, equivalent to a concentration of 220-310 micrograms/g. The pharmacokinetic model proved useful in estimating the total effective dose and concentration of HCB in the litter via the dam's experimental dose. The model was also able to calculate the equivalent human exposure, in order to compare it with actual HCB levels from human monitoring data.

Adolescent↗
PubMed1986

Toxicity of Thermopsis montana in cattle.

Cattle had severe signs of toxicosis when gavaged dried ground Thermopsis montana (false lupine, poison bean, mountain thermopsis) at doses of 0.6-2.8 g/kg/day in a water suspension. Signs included depression, anorexia, swollen eye lids, arched back, tucked abdomen, rough hair coat, and in extremis a prolonged recumbency lasting up to 9 days. Plant potency varied among collections. Total alkaloid doses in collections eliciting severe signs varied from 1.1-11.3 mg/kg/day. There were 5 major alkaloids in each collection that varied in concentration among the collections perhaps accounting for variation in severity of signs elicited. Four of the alkaloids were identified by GC retention time, MS fragmentation patterns and OR analysis as N-methylcytisine, cytisine, (-)-thermopsine, and (-)-anagyrine. Measurements showed a very marked increase of 10X-20X in levels of certain serum enzymes--SGOT, CPK, and LDH that persisted during the period of maximum clinical signs.

Alkaloids↗
PubMed1986

Toxicosis from and possible adaptation to Galega officinalis in sheep and the relationship to Verbesina encelioides toxicosis.

Galega officinalis L (goatsrue), a plant introduced from Europe and found in abundance in northern Utah, was tested for toxicity in ewes (Ovis aries). Clinical signs of poisoning induced by doses as small as 0.8 g of dried plant/kg body weight/day included dyspnea, anoxia, and foaming nasal discharge. Pathologic signs in animals that died following overdose of the plant included severe hydrothorax, generalized lung congestion, foamy exudate in bronchioles and trachea, epicardial and endocardial petechiation, and pericardial effusion in severely affected ewes. Microscopic lesions included a severe diffuse alveolar and interlobular edema. No significant alterations in blood constituents were observed in treated animals. Ewes administered the plant on several consecutive days developed an apparent adaptation to the toxin of the plant and were thereby able subsequently to tolerate levels 5-10 times the pre-treatment lethal dose with no discernible adverse clinical or pathological effects.

Animals↗