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Biomedical subjects

A E Johnson

Publications and source records attributed to A E Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1997

The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane.

Eukaryotic secretory proteins are cotranslationally translocated through the endoplasmic reticulum (ER) membrane via aqueous pores that span the lipid bilayer. Fluorescent probes were incorporated into nascent secretory proteins using modified Lys-tRNAs, and the resulting nascent chains were sealed off from the cytosol in fully assembled translocation intermediates. Fluorescence quenching agents of different sizes were then introduced into the ER lumen in order to determine which were small enough to enter the pore and to quench the fluorescence of probes inside the ribosome and/or the pore. These accessibility studies showed that the aqueous pore in a functioning translocon is 40-60 A in diameter, making it the largest hole observed to date in a membrane that must maintain a permeability barrier.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Cholecystokinin, dopamine D2 and N-methyl-D-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior.

Receptor autoradiography was used to investigate the distribution of brainstem binding sites for cholecystokinin, dopamine and N-methyl-D-aspartate with particular reference to the nucleus of the solitary tract of the rat, an area involved in the control of ingestive behavior. Binding sites for the A and B subtypes of the cholecystokinin receptor, labeled with [(125)I]cholecystokinin octapeptide sulfate in the presence or absence of antagonists for the devazepide (A) or L-365,260 (B) receptor, were present throughout the caudal rostral extent of the nucleus of the solitary tract, the A type predominating in the commissural, medial and gelatinous part and the B type in the lateral part. In the most rostral part of the medial nucleus of the solitary tract, both A and B receptors were present. Dopamine D2 receptors, labeled with [(125)I]NCQ-298, were found in all parts of the nucleus of the solitary tract. No binding to the dopamine D1 receptor, labeled with [(125)I]SCH-23982, was found in the brainstem. N-Methyl-D-aspartate receptors, labeled with [(3)H]dizocilpine maleate, were also present in the entire caudorostral extent of the nucleus of the solitary tract. Binding to cholecystokinin A receptors was co-distributed with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the caudal and rostral parts of the nucleus of the solitary tract, and binding to cholecystokinin B receptors overlapped with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the rostral nucleus of the solitary tract. These results are consistent with the hypothesis that cholecystokinin, dopamine and glutamate interact in the nucleus of the solitary tract in the control of ingestive behavior.

Animals↗
PubMed1997

Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the organization of limbic and motor circuits of the entopeduncular nucleus, suggesting that GABAergic transmission through the limbic pathway is regulated predominantly by glutamic acid decarboxylase-65, whereas glutamic acid decarboxylase-67 is of principal importance in the motor pathway. These data provide additional evidence that the neurons of the limbic and motor subregions of the entopeduncular nucleus are neurochemically distinct.

Animals↗
PubMed1997

Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

Alleles↗
PubMed1997

Factor VIIa-tissue factor: functional importance of protein-membrane interactions.

The first enzyme in the blood clotting cascade consists of two distinct protein subunits: a catalytic subunit (factor VIIa; FVIIa) and an essential regulatory subunit (tissue factor; TF). FVIIa is a soluble plasma protease, while TF is a cell-surface, integral-membrane protein. The recently reported X-ray crystal structure of the complex of FVIIa and the isolated extracellular domain of TF has provided important insights into the protein-protein interactions that bind these two subunits together (1). Equally important in the functioning of the TF-FVIIa complex, but much less well understood, are a series of protein-phospholipid interactions involving TF, FVIIa, and the natural substrates of this enzyme, as well as protein-protein interactions important in substrate recognition by TF-FVIIa. Here we review recent studies on the membrane organization and role of protein-phospholipid interactions in the function of TF-FVIIa, the enzyme that triggers blood clotting in hemostasis and thrombosis.

Crystallography, X-Ray↗
PubMed1996

The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study.

The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-L-arginyl-fVIIa (Fl-FPR-fVIIa). The location of the active-site probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserine-dependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 A assuming a random orientation of donor and acceptor dyes (kappa2 = 2/3; the orientational uncertainty totals approximately 10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane. When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 A after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.

Binding Sites↗
PubMed1996

Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: enhancement of p-guanidinobenzoate substrate hydrolysis.

The mechanism by which the protein cofactor, tissue factor, enhances the activity of its cognate serine protease, coagulation factor VIIa (FVIIa), has been studied using the fluorogenic ester substrate 4-methylumbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue factor (sTF; recombinant human tissue factor containing only the extracellular domain). Pre-steady-state techniques allowed the determination of the individual rate constants for acylation (k2) and deacylation (k3) of the sTF.FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods were required for determination of these parameters for free FVIIa due to extremely slow hydrolysis of MUGB in the absence of sTF. Under all experimental conditions, deacylation was found to be rate-limiting. The major effect of sTF was to raise the affinity of FVIIa for MUGB (31-fold at pH 8.4 and 36-fold at pH 7.6); only minor changes in k2 and k3 were observed. Thus, we conclude that for the ester substrate MUGB, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.

Allosteric Regulation↗
PubMed1996

The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process.

During the cotranslational integration of a nascent protein into the endoplasmic reticulum membrane, the transmembrane (TM) sequence moves out of an aqueous pore formed by Sec61alpha, TRAM, and other proteins and into the nonpolar lipid bilayer. Photocross-linking reveals that this movement involves the sequential passage of the TM domain through three different proteinaceous environments: one adjacent to Sec61alpha and TRAM and two adjacent to TRAM that place different restrictions on TM domain movement. In addition, the TM sequence is not allowed to diffuse into the bilayer from the final TRAM-proximal site until translation terminates. Cotranslational integration is therefore linked to translation and occurs via an ordered multistep pathway at an endoplasmic reticulum site that is multilayered both structurally and functionally.

Cross-Linking Reagents↗
PubMed1996

Correlates of HIV-1 seropositivity among young men in Thailand.

Geographic and demographic correlates of risk for HIV-1 seropositivity were studied in 120,216 young men selected by lottery for service in the Royal Thai Army (RTA). The study population consisted of men selected between November 1991 and May 1993. Venous blood was collected at induction, and a brief demographic questionnaire was administered. HIV-1 seropositivity was established by Western blot confirmation of duplicate reactive ELISAs. Geographic variable provided the strongest correlate of risk, clearly distinguishing residents of the upper north, Bangkok, and the central region from the northeast. Overall 12.2% of men from the upper north were HIV-positive. Men who had lived in rural areas were at less risk in most regions of the country, but had equal risk in the upper north. Unmarried men and those with less education were at higher risk throughout the country. These data provide valuable information on the prevalence of HIV infection in one segment of the general population. Continued surveillance of this group will facilitate evaluation of Thailand's response to the epidemic.

Adult↗
PubMed1996

Undiagnosed systemic lupus erythematosus in the community.

BACKGROUND: Systemic lupus erythematosus (SLE) produces a wide range of symptoms, some of them mild; therefore it may be underdiagnosed. In a pilot study we tested the ability of a screening questionnaire to identify undiagnosed cases in the community. METHODS: A 10-item questionnaire was mailed to 3500 women aged 18-65 randomly selected from a general practice list in Birmingham, UK. A random sample of 300 non-respondents were sent two further mailings. Women returning the questionnaire with three positive answers were to attend for an antinuclear antibody (ANA) test; and those testing positive were to be referred to a rheumatologist for standard clinical assessment. FINDINGS: 1153 (30%) of the women responded, and 307 of these gave three or more positive answers on the questionnaire. The survey disclosed three women with previously undiagnosed SLE; and, in addition, it picked up all four of the women whose SLE had been diagnosed previously. INTERPRETATION: The prevalence of SLE revealed by this survey in Birmingham women is 200/100,000 (95% confidence interval 80 to 412), whereas the prevalence of diagnosed SLE in this age group is 54/100,000 (95% CI 47-62). Thus, a substantial number of undiagnosed cases exist, and further work needs to focus on the prevalence in different ethnic groups and the benefits of early diagnosis and treatment.

Adolescent↗
PubMed1996

Air movement around a worker in a low-speed flow field.

A knowledge of the air movement around a worker in a low-speed airflow is important in a number of areas: containment testing of fume cupboards; testing of personal dust samplers; testing of LEV effectiveness; and measurement of worker exposure. Measurements of velocity vectors around the upper torsos of manikins and a human in low-speed airflows have been made using a laser Doppler anemometer. Both heated and unheated manikins, as well as a 'breathing' manikin were used. The results show that quite distinctive flow patterns develop with heated and unheated bodies. Comparison of the flows around two- and three-dimensional manikins with that around a human shows that only a three-dimensional heated manikin gives good results. The unheated breathing manikin gave results which were unrepresentative of the real situation. A suitable manikin for use in sampling or testing in low-speed airflows would have a heated, rounded, three-dimensional body of reasonably human dimensions and would be non-breathing and clothed.

Air Movements↗
PubMed1996

Integrating culture and healing. Meeting the health care needs of a multicultural community.

Delivering health care to culturally diverse patients is fast becoming an integral part of patient care-a change driven by shifting demographics in Minnesota and especially in the Twin Cities metro area. At United Hospital and Children's Health Care-St. Paul, ethnographic research is being used to create cross-cultural health care information systems that address the needs of providers and patients. These include an easy-to-use computer-based information system, brown bag seminars, and cross-cultural skills training. This article discusses that hospitals' efforts to identify provider needs, collect cultural information, and disseminate that information in a manner that supports quality and cost-effective health care delivery.

Adult↗
PubMed1996

The translocon: more than a hole in the ER membrane?

In eukaryotes, the vast majority of secreted and integral membrane proteins are targeted to the membrane of the endoplasmic reticulum (ER) early during translation. These polypeptides are then either transported across or inserted into the ER membrane at sites termed translocons. As protein translocation occurs through an aqueous pore, the minimal requirement for a translocon is a passive structure that provides a passage-way across the membrane. However, recent data suggest that the translocon is a complex structure that orchestrates the localization, orientation, maturation and possibly degradation of nascent chains.

Biological Transport↗
PubMed1995

Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.

The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Transfer↗
PubMed1995

Neuroleptics differentially modulate central dopamine D1-receptor binding.

The effect of the classical neuroleptic, fluphenazine, on dopamine D1-receptor binding was examined in different regions of the basal ganglia. Whereas exposure to fluphenazine for 18 months reduced [125I]SCH-23982 binding to D1-receptors in the caudate putamen, nucleus accumbens and olfactory tubercle, binding in the entopeduncular nucleus was enhanced after fluphenazine treatment. Competition studies indicated that the region-dependent changes in [125I]SCH-23982 binding after fluphenazine exposure were not due to differences in the affinity of fluphenazine or other dopamine ligands for D1-binding sites. These data suggest that in addition to modulating striatal function, classical neuroleptics may also alter neurotransmission in the basal ganglia by enhancing dopamine receptor binding in the entopeduncular nucleus.

Animals↗
PubMed1995

Castration reduces vasopressin receptor binding in the hamster hypothalamus.

A recently developed ligand with very high affinity and selectivity for the vasopressin (AVP) V1a receptor subtype (i.e. [125I]Linear AVP antagonist ([125I]-LinAntag) was used to describe the distribution of AVP binding sites in the hamster brain, and to determine whether AVP receptor binding was influenced by testicular hormones in sites involved in the regulation of steroid-dependent social behaviors. These studies demonstrated [125I]LinAntag binding in regions of the hamster brain which have not been previously identified with other AVP ligands. In addition, testicular hormones were found to alter [125I]LinAntag binding in two distinct regions, the posterior lateral preoptic-anterior lateral hypothalamic continuum and the posterior ventrolateral hypothalamic nucleus and adjacent tuberal area.

Animals↗
PubMed1995

The prevalence and incidence of systemic lupus erythematosus in Birmingham, England. Relationship to ethnicity and country of birth.

OBJECTIVE: To establish the point prevalence of systemic lupus erythematosus (SLE) on January 1, 1992, and the incidence of SLE during 1991, in Birmingham, England, a large city with a broad ethnic mix. METHODS: Six sources were used to ascertain patients with diagnosed SLE, including notification by attending and primary care physicians, the lupus patient support group, and hospital inpatient and laboratory data. RESULTS: There were 242 SLE patients (227 females, 15 males) identified: prevalence rate 27.7/100,000 (95% confidence interval 24.2-31.2/100,000) in the population and 206.0/100,000 in Afro-Caribbean females. No significant differences in female ethnic prevalence rates by place of birth were observed. Thirty-three patients developed SLE in 1991: incidence rate 3.8/100,000/year (95% confidence interval 2.5-5.1/100,000/year). CONCLUSION: This study illustrates dramatic differences in incidence and prevalence rates in the UK, depending on ethnic group and irrespective of place of birth.

Adolescent↗