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Biomedical subjects

R S Johnson

Publications and source records attributed to R S Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1993

Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.

Profilaggrin is an intermediate filament-associated protein of cornified epithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisional activation of multiply charged precursor ions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-termini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino- and carboxyl-termini were "ragged", suggesting that processing may involve exopeptidase action after an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.

Amino Acid Sequence↗
PubMed1993

Targeted disruption of CD43 gene enhances T lymphocyte adhesion.

CD43 is a major leukocyte surface glycoprotein thought to have important functions for T lymphocyte adhesion and activation. We investigated the function of CD43 by using gene targeting to eliminate CD43 expression in the human T lymphocyte line CEM and then testing their adhesive phenotype. Loss of CD43 expression by the CEM cells enhanced their homotypic adhesion and binding to two distinct ligands, fibronectin and HIV-1 gp120. The enhanced homotypic adhesion was blocked specifically by an anti-beta 1 integrin mAb, and the enhanced binding to fibronectin and gp120 was blocked specifically by anti-beta 1 integrin and anti-CD4 mAb, respectively. Partial reconstitution of CD43 expression in the CD43-negative cells resulted in a corresponding reversion to a less adhesive phenotype. These data suggest that CD43 interferes with T lymphocyte adhesion and that CD43 can regulate lymphocyte adhesion by providing a threshold that must be overcome for cell-cell and cell-ligand interactions to occur.

Antigens, CD↗
PubMed1993

Sequential phosphorylation of rhodopsin at multiple sites.

Photolyzed rhodopsin is phosphorylated at multiple serine and threonine residues during the quenching of phototransduction. Sites of phosphorylation by rhodopsin kinase have been localized to the C-terminal region of rhodopsin, but no information was available on the kinetics and identity of phosphorylated residues. To determine the kinetics of phosphorylation at specific residues, the phosphorylated C-terminal peptide of rhodopsin (330DDEASTTVSKTETSQVAPA) obtained by proteolysis of rhodopsin with endoproteinase Asp-N was subjected to further subdigestion followed by electrospray mass spectrometry. Analysis of monophosphorylated peptide revealed that the major initial phosphorylation site is 338Ser. The analysis of di- and triphosphorylated peptides indicated that 343Ser or 336Thr residues are subsequent phosphorylation sites. These three residues, located in the C-terminal region of rhodopsin, are likely to be key phosphorylation sites of rhodopsin during the quenching of phototransduction. Identification of the kinetics of phosphorylation will facilitate understanding the functional significance of rhodopsin phosphorylation at multiple sites and the mechanism of rhodopsin kinase action.

Amino Acid Sequence↗
PubMed1993

Mice lacking major histocompatibility complex class I and class II molecules.

Mice lacking major histocompatibility complex (MHC) antigens were generated by mating beta 2-microglobulin-deficient, and therefore class I-deficient, animals with MHC class II-deficient animals. When housed under sterile conditions, the resulting MHC-deficient mice appear healthy, survive for many months, and breed successfully. Phenotypically, MHC-deficient mice are depleted of CD4+ and CD8+ T cells in peripheral lymphoid organs due to a lack of appropriate restricting elements. In contrast, the B-cell compartment of these animals appears intact, and MHC-deficient mice can mount specific antibody responses when challenged with a T-independent antigen. Spleen cells from MHC-deficient animals are poor stimulators and responders in a mixed lymphocyte reaction. Despite their relatively weak cellular immune responses in vitro, MHC-deficient mice reject allogeneic skin grafts with little delay, and grafts from MHC-deficient animals are rapidly rejected by normal allogeneic recipients. Taken together, these results emphasize the plasticity of the immune system and suggest that MHC-deficient mice may be useful for examining compensatory mechanisms in severely immunocompromised animals.

Animals↗
PubMed1993

Relaxation for insomnia and hypnotic medication use in older women.

Relaxation therapy was given to 3 groups of older women (N = 57): (a) hypnotically medicated insomniacs, (b) nonhypnotically medicated insomniacs, and (c) noninsomniacs. Groups b and c were receiving antihypertensives. Self-reported sleep and medication data were collected for 1 week at pretreatment (except relaxation), posttreatment, and 6-weeks follow-up. Three relaxation sessions, a nondemanding, hybrid method, were administered with the rationale of helping insomnia or high blood pressure. Substantial sleep improvement occurred only for nonhypnotically medicated insomniacs. Substantial sleep medication reduction (47%) occurred only for hypnotically medicated insomniacs. This relaxation approach proved valuable, but the nature of the treatment effect was dependent on the medication status of the insomniac.

Aged↗
PubMed1993

A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture.

The AP-1 transcription factors are considered immediate-early response genes and are thought to be involved in a wide range of transcriptional regulatory processes linked to cellular proliferation and differentiation. To study one of the key members of this family, the proto-oncogene c-jun, we have used homologous recombination-mediated gene targeting to produce mice with a c-jun null mutation. c-jun null embryos die at mid-gestation, with an average time of death of 12.5 days postcoitus. Homozygous mutant embryos are indistinguishable from wild-type littermates both grossly and histologically until the time of death. However, primary fibroblasts derived from live heterozygous and homozygous mutant embryos show greatly reduced growth rates in culture. The subnormal mitogenic response of these cells cannot be overcome by the addition of a number of purified mitogens. These studies indicate that although c-jun is not required for cellular proliferation and differentiation up to mid-gestation, it is required for survival past that stage as well as for the mitogenic response of embryonic fibroblasts in culture.

Animals↗
PubMed1992

Pleiotropic effects of a null mutation in the c-fos proto-oncogene.

The c-fos proto-oncogene has been implicated as a central regulatory component of the nuclear response to mitogens and other extracellular stimuli. Embryonic stem cells targeted at the c-fos locus have been used to generate chimeric mice that have transmitted the mutated allele through the germline. Homozygous mutants show reduced placental and fetal weights and significant loss of viability at birth. Approximately 40% of the homozygous mutants survive and grow at normal rates until severe osteopetrosis, characterized by foreshortening of the long bones, ossification of the marrow space, and absence of tooth eruption, begins to develop at approximately 11 days. Among other abnormalities, these mice show delayed or absent gametogenesis, lymphopenia, and altered behavior. Despite these defects, many live as long as their wild-type or heterozygous littermates (currently 7 months). These data indicate that c-fos is not required for the growth of most cell types but is involved in the development and function of several distinct tissues.

Animals↗
PubMed1992

Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.

The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes.

Alleles↗
PubMed1992

The rod transducin alpha subunit amino terminus is heterogeneously fatty acylated.

Rod transducin (Tr), a heterotrimeric GTP-binding protein composed of alpha, beta, and gamma subunits, couples photolysis of rhodopsin to the activation of cyclic GMP phosphodiesterase in the vertebrate visual signal transduction cascade. To determine if T alpha r is covalently modified, we analyzed tryptic fragments of bovine retinal T alpha r using electrospray mass spectrometry, liquid chromatography/mass spectrometry, tandem mass spectrometry, and gas chromatography. A novel heterogeneous fatty acylation was detected at the NH2 terminus. Four types of NH2-terminal tryptic fragments of T alpha r were isolated, and each contained either a lauroyl (C12:0), myristoyl (C14:0), (cis-delta 5)-tetradecaenoyl (C14:1) or (cis,cis-delta 5, delta 8)-tetradecadienoyl (C14:2) fatty acyl residue amide-linked to the NH2-terminal glycine residue. NH2-terminal fatty acylation does not anchor T alpha r permanently in the membrane, since T alpha r used in these experiments was eluted without detergent from rod outer segment membranes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

The NH2 terminus of retinal recoverin is acylated by a small family of fatty acids.

Recoverin is a recently identified Ca(2+)-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.

Acylation↗
PubMed1992

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.

Animals↗
PubMed1992

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.

Algorithms↗
PubMed1992

Are insomniacs sleepy during the day? A pupillometric assessment.

A biodevelopmental model of insomnia is articulated specifying coordinated nighttime (disturbed sleep pattern) and daytime (no excessive daytime sleepiness) characteristics defining an insomnoid classification in at-risk groups: short sleepers and older adults. Pupillometry is proposed as a useful means of discriminating degree of daytime sleepiness to aid in the differential diagnosis of insomnia and insomnoid states, and the present study tested the discriminative validity of this approach. Noninsomniac (n = 34) and insomniac (n = 29) college students submitted to four 10 min pupillometry sessions tracking daytime sleepiness from morning arising to bedtime. Pupil diameter proved to be an able discriminator of these two groups though substantial overlap of the two distributions was also noted. The results supported the sensitivity of pupillometry in detecting daytime sleepiness, but yielded alternative interpretations. We observed statistical differentiation in insomniac and noninsomniac daytime sleepiness, but substantial, functional overlap between these groups. Assessment and treatment implications arising from the biodevelopmental model were hypothesized.

Adolescent↗
PubMed1992

Personality characteristics of cocaine and alcohol abusers: more alike than different.

Although there is extensive literature describing the characteristics of individuals entering treatment for alcoholism, few research reports are available describing the personality characteristics of individuals addicted to cocaine. Forty-four matched patient pairs, one member of each pair who abused cocaine and the other alcohol, were compared on the basis of their Minnesota Multiphasic Personality Inventory (MMPI) profiles. The subjects were matched on the basis of age, sex, and race from a larger population entering an inpatient addiction treatment center. Statistical analysis revealed more similarities than differences between the two groups, with the average profile type for both cocaine and alcohol abusers being 4-2. These results are discussed in the context of previous reports of psychopathology among cocaine abusers and personality theories of drug choice. The number of different code types within each substance abuse group is also reported and the concept of profile heterogeneity supported. Profile subtypes among cocaine patients are described clinically along with their implications for treatment.

Adolescent↗
PubMed1991

Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice.

The maturation of T cells in the thymus is dependent on the expression of major histocompatibility complex (MHC) molecules. By disruption of the MHC class II Ab beta gene in embryonic stem cells, mice were generated that lack cell surface expression of class II molecules. These MHC class II-deficient mice were depleted of mature CD4+ T cells and were deficient in cell-mediated immune responses. These results provide genetic evidence that class II molecules are required for the maturation and function of mature CD4+ T cells.

Animals↗
PubMed1991

Preparation and characterization of N-(1-pyrenyl)iodoacetamide-labeled Escherichia coli RNA polymerase.

N-(1-Pyrenyl)iodoacetamide has been used to introduce fluorescent probes into Escherichia coli RNA polymerase. After an incubation time of 15 min, approximately 2 pyrene equiv was introduced per enzyme molecule. There was no further increase in modification after more extended periods of incubation. Neither calf thymus DNA nor nucleotides protected the holoenzyme from modification. Thus, the sites of modification do not appear to involve the binding sites for polynucleotides or the ribonucleoside triphosphates. From the isolation and analysis of the individual subunits, it was found that sigma contained approximately 1 pyrene equiv, beta contained 0.6, beta' contained 0.6, and alpha less than 0.1. Spectral and Stern-Volmer analyses indicate that the covalently attached pyrene molecules are in comparable apolar microenvironments. On the basis of CD analyses, the introduction of pyrene molecules into RNA polymerase alters its secondary structure. This alteration in secondary structure manifests itself by a reduction in overall enzymatic activity. Transcript analysis of the products obtained by using a linearized plasmid containing the A1 promoter and the Te terminator of bacteriophage T7 indicates that the pyrenyl derivative is capable of producing full-length transcripts and that it has an efficiency of chain termination comparable to the native enzyme. Analysis of tau plots for the interaction of the pyrenyl derivative and the native enzyme, respectively, with the A1 promoter yielded comparable values for the isomerization constant in the conversion of the closed complex to an open one. Comparable values were also obtained for the association constant. The rate of chain elongation for the pyrenyl derivative, however, is approximately 54% of that observed for the native enzyme. Thus, the decrease in overall transcriptional activity observed with the pyrenyl derivative is not due to a decrease in the efficiency of initiation or premature termination, but rather to a decrease in the rate of chain elongation.

Circular Dichroism↗
PubMed1991

Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy.

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-Directed RNA Polymerases↗