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J E Johnson

Publications and source records attributed to J E Johnson.

At least 127 records · Page 7Linked to original sources
PubMed1998

Lipid structure and not membrane structure is the major determinant in the regulation of protein kinase C by phosphatidylserine.

This study addresses the molecular basis for protein kinase C's specific activation by phosphatidylserine. Specifically, we ask whether protein kinase C's phospholipid specificity arises from specific protein/lipid interactions or whether it arises from unique membrane-structuring properties of phosphatidylserine. We measured the interaction of protein kinase C betaII to membranes that differed only in being enantiomers to one another: physical properties such as acyl chain composition, membrane fluidity, surface curvature, microdomains, headgroup packing, and H-bonding with water were identical. Binding and activity measurements reveal that protein kinase C specifically recognizes 1, 2-sn-phosphatidyl-L-serine, independently of membrane structure. High-affinity binding and activation are abolished in the presence of enantiomeric membranes containing 2,3-sn-phosphatidyl-L-serine, 2, 3-sn-diacylglycerol, and 2,3-sn-phosphatidylcholine. Our data also show that the stereoselectivity for 1,2-sn-diacylglycerol is not absolute; 2,3-sn-diacylglycerol modestly increases the membrane affinity of protein kinase C provided that 1, 2-sn-phosphatidyl-L-serine is present. We also find that the stereochemistry of the bulk phospholipid, in this case phosphatidylcholine, has no significant influence on protein kinase C's membrane interaction. These data reveal that specific molecular determinants on protein kinase C stereospecifically recognize structural determinants of phosphatidylserine.

Binding Sites↗
PubMed1998

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase.

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).

Amino Acid Sequence↗
PubMed1998

Multiple elements regulate Mash1 expression in the developing CNS.

Mash1, a transcription factor of the basic helix-loop-helix class, is expressed during embryogenesis in restricted regions of the nervous system. An essential role for Mash1 in neural development was demonstrated previously in mice carrying a targeted disruption of the Mash1 gene. Regulation of the precise temporal and spatial expression of Mash1 is thus likely to be important for proper neural development. In this study, sequences that regulate Mash1 expression in the central nervous system were characterized by assaying the expression of lacZ reporter genes in transgenic embryos. A 1158-bp enhancer localized approximately 7 kb upstream of the Mash1 coding region was identified. Deletions within this enhancer region reveal the presence of both positive and negative cis-acting elements. Analysis of multiple sequences within the enhancer demonstrate that different elements preferentially function in different regions within the Mash1-specific CNS expression domain. In addition, a role for sequences 3' of the Mash1 coding region is revealed, providing evidence for posttranscriptional control of Mash1 expression in multiple CNS domains.

Animals↗
PubMed1998

Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryoelectron microscopy and image reconstruction.

Cryoelectron microscopy and three-dimensional image reconstruction analysis has been used to determine the structure of native and in vitro assembled cowpea chlorotic mottle virus (CCMV) virions and capsids to 25-A resolution. Purified CCMV coat protein was used in conjunction with in vitro transcribed viral RNAs to assemble RNA 1 only, RNA 2 only, RNA 3/4 only, and empty (RNA lacking) virions. The image reconstructions demonstrate that the in vitro assembled CCMV virions are morphologically indistinguishable from native virions purified from infected plants. The viral RNA (vRNA) is packaged similarly within the different types of virions. The centers of all assembled particles are generally devoid of density and the vRNA packs against the interior surface of the virion shell. The vRNA appears to adopt an ordered conformation at each of the quasi-threefold axes.

Bromovirus↗
PubMed1998

Crystallization and preliminary X-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid.

HK97 is a temperate dsDNA bacteriophage of Escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 A using synchrotron radiation. The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution. A capsid precursor (prohead) was made in vivo by expressing capsid proteins in E. coli. This prohead was purified, converted to Head II in vitro, and used to grow crystals. The empty Head II has the same form as the mature HK97 capsid, but without DNA. The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling. The unit cell is monoclinic, with dimensions a = 580 A, b = 625 A, c = 790 A, beta = 90.0 degrees and two particles per unit cell.

Bacteriophages↗
PubMed1998

BDNF injected into the superior colliculus reduces developmental retinal ganglion cell death.

The role of neurotrophins as survival factors for developing CNS neurons, including retinal ganglion cells (RGCs), is uncertain. Null mutations for brain-derived neurotrophic factor (BDNF) or neurotrophin 4 (NT4), individually or together, are without apparent effect on the number of RGCs that survive beyond the period of normal, developmental RGC death. This contrasts with the BDNF dependence of RGCs in vitro and the effectiveness of BDNF in reducing RGC loss after axotomy. To investigate the effect of target-derived neurotrophins on the survival of developing RGCs, we injected BDNF into the superior colliculus (SC) of neonatal hamsters. At the age when the rate of developmental RGC death is greatest, BDNF produces, 20 hr after injection, a 13-15-fold reduction in the rate of RGC pyknosis compared with the rates in vehicle-injected and untreated hamsters. There is no effect 8 hr after injection. Electrochemiluminescence immunoassay measurements of BDNF protein in the retinae and SC of normal and BDNF-treated hamsters demonstrate that the time course of BDNF transport to RGCs supports a role for target-derived BDNF in promoting RGC survival. The effectiveness of pharmacological doses of BDNF in reducing developmental RGC death may be useful in further studies of the mechanisms of stabilization and elimination of immature central neurons.

Amidines↗
PubMed1998

The structure of tobacco ringspot virus: a link in the evolution of icosahedral capsids in the picornavirus superfamily.

BACKGROUND: Tobacco ringspot virus (TRSV) is a member of the nepovirus genus of icosahedral RNA plant viruses that cause disease in fruit crops. Nepoviruses, comoviruses and picornaviruses are classified in the picornavirus superfamily. Crystal structures of comoviruses and picornaviruses and the molecular mass of the TRSV subunit (sufficient to accommodate three beta-barrel domains) suggested that nepoviruses may represent a link in the evolution of the picornavirus capsids from a T = 3 icosahedral virus. This evolutionary process is thought to involve triplication of the capsid protein gene, to encode a three-domain polyprotein, followed by development of cleavage sites in the interdomain linking regions. Structural studies on TRSV were initiated to determine if the TRSV subunit corresponds to the proposed uncleaved three-domain polyprotein. RESULTS: The 3.5 A resolution structure of TRSV shows that the capsid protein consists of three beta-barrel domains covalently linked by extended polypeptides. The order of connectivity of the domains in TRSV confirms the proposed connectivity for the precleaved comovirus and picornavirus capsid polyprotein. Structural differences between equivalent domains in TRSV and comoviruses are confined to the external surface loops, interdomain connecting polypeptides and N termini. The three different domains within TRSV and comoviruses are more closely related at the structural level than the three individual domains within picornaviruses. CONCLUSIONS: The structural results confirm the notion of divergent evolution of the capsid polyproteins of nepoviruses, comoviruses and picornaviruses from a common ancestor. A number of residues were found to be conserved among various nepoviruses, some of which stabilize the quaternary structure of the three domains in the TRSV capsid protein subunit. Two conserved regions were identified on the external surface of TRSV, however, mutational studies will be needed to understand their functional significance. Nepoviruses transmitted by the same nematode species do not share regions with similar amino acid composition on the viral surface.

Amino Acid Sequence↗
PubMed1998

Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry.

Virus particles are stable yet exhibit highly dynamic character given the events that shape their life cycle. Isolated from their hosts, the nucleoprotein particles are macromolecules that can be crystallized and studied by x-ray diffraction. During assembly, maturation and entry, however, they are highly dynamic and display remarkable plasticity. These dynamic properties can only be inferred from the x-ray structure and must be studied by methods that are sensitive to mobility. We have used matrix-assisted laser desorption/ionization mass spectrometry combined with time resolved, limited proteolysis (Cohen, S. L., Ferre-D'Amare, A. R., Burley, S. K., and Chait, B. T. (1995) Protein Sci. 4, 1088-1099; Kriwacki, R. W., Wu, J., Tennant, T., Wright, P. E., and Siuzdak, G. (1997) J. Chromatogr. 777, 23-30; Kriwacki, R. W., Wu, J., Siuzdak, G., and Wright, P. E. (1996) J. Am. Chem. Soc. 118, 5320-5321) to examine the viral capsid of flock house virus. Employing less than 10 microg of virus, time course digestion products were assigned to polypeptides of the subunit. Although surface regions in the three-dimensional structure were susceptible to cleavage on extended exposure to the protease, the first digestion products were invariably from parts of the subunit that are internal to the x-ray structure. Regions in the N- and C-terminal portions of the subunit, located within the shell in the x-ray structure, but implicated in RNA neutralization and RNA release and delivery, respectively, were the most susceptible to cleavage demonstrating transient exposure of these polypeptides to the viral surface.

Animals↗
PubMed1998

Molecular packing in virus crystals: geometry, chemistry, and biology.

An automated procedure was developed to determine the geometrical and chemical interactions of crystalline virus particles using the crystal parameters, particle position, orientation, and atomic coordinates for an icosahedral asymmetric unit. Two applications of the program are reported: (1) An analysis of a novel pseudo-rhombohedral (R32) symmetry present in the monoclinic crystal lattices of both Nodamura Virus (NOV) and Coxsackie virus B3 (CVB3). The study shows that in both cases the interactions between particles is substantially increased by minor deviations from exact R32 symmetry and that only particles with the proper ratio of dimensions along twofold and fivefold symmetry axes (such as southern bean mosaic virus) can achieve comparable buried surface area in the true R32 space group. (2) An attempt was made to correlate biological function with remarkably conserved interparticle contact regions found in different crystal forms of three members of the nodavirus family, NOV, Flock House Virus (FHV), and Black Beetle Virus (BBV). Mutational evidence implicates the quasi-threefold region on the viral surface in receptor binding in nodaviruses and this region is dominant in particle contacts in all three virus crystals. Examination of particle contacts in numerous crystal structures of viruses in the picornavirus super-family showed that portions of the capsid surface known to interact with a receptor or serve as an epitope for monoclonal antibodies frequently stabilize crystal contacts.

Computer Simulation↗
PubMed1998

Energetics of quasiequivalence: computational analysis of protein-protein interactions in icosahedral viruses.

Quaternary structure polymorphism found in quasiequivalent virus capsids provides a static framework for studying the dynamics of protein interactions. The same protein subunits are found in different structural environments within these particles, and in some cases, the molecular switching required for the polymorphic quaternary interactions is obvious from high-resolution crystallographic studies. Employing atomic resolution structures, molecular mechanics, and continuum electrostatic methods, we have computed association energies for unique subunit interfaces of three icosahedral viruses, black beetle virus, southern bean virus, and human rhinovirus 14. To quantify the chemical determinants of quasiequivalence, the energetic contributions of individual residues forming quasiequivalent interfaces were calculated and compared. The potential significance of the differences in stabilities at quasiequivalent interfaces was then explored with the combinatorial assembly approach. The analysis shows that the unique association energies computed for each virus serve as a sensitive basis set that may determine distinct intermediates and pathways of virus capsid assembly. The pathways for the quasiequivalent viruses displayed isoenergetic oligomers at specific points, suggesting that these may determine the quaternary structure polymorphism required for the assembly of a quasiequivalent particle.

Animals↗
PubMed1998

Frequency of parvovirus B19 infection in nonimmune hydrops fetalis and utility of three diagnostic methods.

The rate of parvovirus B19 (PV) infection in cases of "idiopathic" nonimmune hydrops fetalis (NIHF) is reported to be approximately 16% with polymerase chain reaction (PCR)-based methods. Antibodies for use in paraffin-embedded tissue have not been systematically compared with PCR or with the presence of inclusions at varying gestational ages. All autopsy cases of NIHF and those with effusions of multiple serous membranes examined between 1991 and 1996 (n = 29) were evaluated for the presence of PV DNA by PCR analysis of paraffin-embedded liver tissue. PCR-positive cases and "idiopathic" cases were examined for the presence of inclusions in routine histological sections and for PV protein using a monoclonal antibody (NovoCastra R92F6). Among the four clinically idiopathic cases, one (25%) was positive for PV using PCR. The three negative idiopathic cases had no inclusions and were negative for PV by PCR and immunohistochemistry (IHC); all were third-trimester gestations (28, 31, and 32 weeks). Identifiable risk factors for NIHF other than PV in the remaining 25 cases included cystic hygroma, seven (three 45,X; two 46,XX; two no growth); complex cardiac anomaly, six; infection, three (two CMV, one chlamydia); twin-twin transfusion, two; lymphangiectasia, two; diaphragmatic hernia, tracheal atresia, trisomy 21, congenital cystic adenomatoid malformation, one each. One of these nonidiopathic cases, a fetus with cystic hygroma and a 45,X karyotype, was positive for PV DNA only on the blot, consistent with a low titer; no inclusions were present, and IHC was negative in multiple organs in this instance. One of four (25%) cases of idiopathic NIHF cases contained PV DNA by PCR analysis; there were abundant inclusions in multiple organs, and IHC was strongly positive as well. Of 25 cases of nonidiopathic NIHF, one (4%) was also positive for PV DNA by PCR. PV protein was detected by IHC only in the presence of inclusions; IHC thus may be useful for highlighting sparse inclusions. No second-trimester case of NIHF was unexplained. Late (third-trimester) cases of "idiopathic" NIHF are likely to be negative by all methods, either because they are not attributable to PV infection or because PV protein and DNA are below detectable levels or are no longer present. Maternal serology for PV and TORCH agents may be the best method for investigating third-trimester losses to otherwise unexplained NIHF.

Adult↗
PubMed1998

The use of solid physical models for the study of macromolecular assembly.

The use of modern technology in the construction of accurate solid macromolecular models based on atomic coordinates and electron density functions has led us to re-examine the usefulness of physical models as tools for understanding molecular assembly and for designing detailed experimental and computational studies of the assembly process. Recent developments include the construction of new models, which have provided insights into the assembly of viruses and light harvesting complexes.

Biomedical Engineering↗
PubMed1998

The University of Alabama Teenage Access Project: a model for prevention, referrals, and linkages to testing for high-risk young women.

The Teenage Access Project (TAP) was supported by the Special Projects of National Significance Program to expand and ensure access to health and support services for disadvantaged, human immunodeficiency virus (HIV)-positive, and at-risk adolescent and young adult women aged 10-21 years by: (a) preventing further HIV transmission through empowerment and reduction of risk behavior, (b) providing HIV counseling and testing to increase screening of young women, and (c) facilitating referrals to medical and psychosocial services through the TAP activities. TAP served 403 young people (82% African-American and 83% young women). The basic elements of the TAP service model components were: (a) outreach to community agencies serving high-risk, disadvantaged young women; (b) My Individual Responsibility Reduces Our Risk (MIRROR), a six-module risk-reduction and empowerment activity specifically designed for young women in Jefferson County, using a small group format; and (c) the Adolescent Testing Center (ATC), a center for age and culturally appropriate HIV testing, pre- and posttest counseling, risk assessment, and referrals. HIV testing and pre- and posttest counseling were provided confidentially in both clinical and community settings. More than 200 participated in the MIRROR activity. A total of 101 young women received HIV counseling and testing services.

AIDS Serodiagnosis↗