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PubMed1994

Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells.

We have previously demonstrated that human papillomavirus type 1 (HPV 1) and 16 (HPV 16) E4 proteins form cytoplasmic filamentous networks which specifically colocalize with cytokeratin intermediate-filament (IF) networks when expressed in simian virus 40-transformed keratinocytes. The HPV 16 (but not the HPV 1) E4 protein induced the collapse of the cytokeratin networks. (S. Roberts, I. Ashmole, G. D. Johnson, J. W. Kreider, and P. H. Gallimore, Virology 197:176-187, 1993). The mode of interaction of E4 with the cytokeratin IFs is unknown. To identify E4 sequences important in mediating this interaction, we have constructed a large panel of mutant HPV (primarily HPV 1) E4 proteins and expressed them by using the same simian virus 40-epithelial expression system. Mutation of HPV 1 E4 residues 10 to 14 (LLGLL) abrogated the formation of cytoplasmic filamentous networks. This sequence corresponds to a conserved motif, LLXLL, found at the N terminus of other E4 proteins, and similar results were obtained on deletion of the HPV 16 motif, LLKLL (residues 12 to 16). Our findings indicate that this conserved motif is likely to play a central role in the association between E4 and the cytokeratins. An HPV 1 E4 mutant protein containing a deletion of residues 110 to 115 induced the collapse of the cytokeratin IFs in a manner analogous to the HPV 16 E4 protein. The sequence deleted, DLDDFC, is highly conserved between cutaneous E4 proteins. HPV 1 E4 residues 42 to 80, which are rich in charged amino acids, appeared to be important in the cytoplasmic localization of E4. In addition, we have mapped the N-terminal residues of HPV 1 E4 16-kDa and 10/11-kDa polypeptides expressed by using the baculovirus system and shown that they begin at tyrosine 16 and alanine 59, respectively. Similar-sized E4 proteins are also found in vivo. N-terminal deletion proteins, which closely resemble the 16-kDa and 10/11-kDa species, expressed in keratinocytes were both cytoplasmic and nuclear but did not form cytoplasmic filamentous networks. These findings support the postulate that N-terminal proteolytic processing of the E1-- E4 protein may modulate its function in vivo.

Adult↗
PubMed1980

Structural features of extraocular muscles of children with strabismus.

Ninety extraocular muscle (EOM) biopsy specimens from 80 children with strabismus and normal ocular alignment were studied by light microscopy, enzyme histochemistry, and electron microscopy. Light microscopy showed considerable variation in muscle fiber shape and size with sarcomere disruption, sharp increases in endomysial and perimysial collagen, numerous vacuoles and subsarcolemmal inclusions. Electron microscopy showed disruption of myofilaments, nemaline rods, abnormal mitochondria, leptomeric profiles, occasional "myelin figures," glycogen, and lipid-like droplets. Some intramuscular nerves contained long-spacing collagen ("Luse bodies"). Unlike somatic skeletal muscle, EOM showed a consistent lack of mosaic pattern and reciprocal stain activity between fiber types noted by enzyme histochemistry. Quantitative observations indicate that minimal but consistent morphologic differences exist between strabismic and nonstrabismic EOM. The role these changes play in the pathogenesis of strabismus and their significance remains unclear.

Adenosine Triphosphatases↗
PubMed1996

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258.

The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.

Base Sequence↗
PubMed2000

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail.

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.

Animals↗
PubMed1977

Fine structural features of adrenergic nerve fibers and endings in the pineal gland of the rat, ground squirrel and chinchilla.

The ultrastructural features of the adrenergic nerve fibers in the pineal glands of the rat, ground squirrel and chinchilla are described. Frequency distribution histograms of diameters of granulated and non-granulated vesicles in the adrenergic nerve endings demonstrate that the pineal nerve endings in the chinchilla contain a considerable number of large granulated and non-granulated vesicles, in contrast to those in the rat and ground squirrel. Synaptic ribbons seen in the pinealocytes of the ground squirrel were often localized near that plasma membrane which lay in close proximity to the axolemma of adrenergic nerve fibers. This observation may indicate that the synaptic ribbons are involved in the functional interconnection between pinealocytes and adrenergic nerve fibers. Localized dilations of the adrenergic nerve fibers were commonly observed in the pineal glands of all species examined. In addition to a variety of axonal constituents, various forms of inclusion bodies were tightly packed within these axonal dilations. The accumulation of the inclusion bodies may represent degenerative changes which occur in the pineal adrenergic nerve fibers in relation to the functional activity of the pineal gland.

Adrenergic Fibers↗
PubMed2003

Fine structural features of the acetylcholine innervation in the developing neostriatum of rat.

The acetylcholine (ACh) innervation in the developing neostriatum of rat was examined by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). ChAT-immunoreactive cell bodies and their emerging processes, located at birth in the lateral part of neostriatum, progressively pervaded the whole region, to give rise to an extremely dense axonal network. As visualized and measured in single thin sections, at postnatal (P) ages P8, P16, and P32, the intrinsic and relational features of ChAT-immunostained profiles of axon varicosities in the lateral and medial parts of neostriatum were similar to those previously described in the adult. At the three postnatal ages, the immunoreactive profiles were comparable in shape, size, and vesicular content, and displayed one or more mitochondria with increasing frequency (from 10% at P8 to 29% at P32). The proportion which showed a synaptic junction was low at the three ages (8-16%), indicating an average synaptic incidence of 22% for whole varicosities after stereological extrapolation. The observed junctions were relatively small, mostly symmetrical, and made with dendritic spines or branches. The frequency of synapses on spines versus branches increased with age, from 20% at P8 to almost 60% at P32. Thus, the relational features of the neostriatal ACh innervation were similar to adult as soon as it appeared, as previously observed to be the case in the developing cerebral cortex. The diffuse mode of transmission may therefore be characteristic of both ACh interneurons (neostriatum) and projection neurons (cerebral cortex) in the CNS, and could be determining their functional properties during development as well as at maturity.

Acetylcholine↗
PubMed1988

Involvement in the initiation of T cell responses and structural features of an 85-kDa membrane activation antigen.

A monoclonal antibody, 7F7, directed at a recently described membrane activation antigen of 85 kDa was found to inhibit the T cell proliferation induced by an anti-CD3 antibody, phytohemagglutinin A and concanavalin A. The T cell response to allogenic stimulator cells was also weakly inhibited. The inhibition of these T cell responses was only obtained if the antibody was added within the first 8 h (first 24 h in the case of the concanavalin A response) of culture. In addition the antibody inhibited the formation of cellular aggregates seen in stimulated cultures when added within the first 8 h. The membrane glycoprotein recognized by 7F7 is shown to have a slightly different molecular mass on cells of different lineage, a protein core of 55 kDa and to contain 30-50% of N-linked as well as a small amount of O-linked carbohydrates and sialic acid residues. This study suggests that the highly glycosylated membrane activation antigen defined by antibody 7F7 could contribute to the contact between those cells which are involved in the initiation of T cell responses.

Antibodies, Monoclonal↗
PubMed1987

Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro.

In vitro, efficient translocation and glycosylation of the precursor of yeast alpha-factor can take place post-translationally. This property of prepro-alpha-factor appears to be unique as it could not be extended to other yeast protein precursors such as preinvertase or preprocarboxypeptidase Y. In order to determine if specific domains of prepro-alpha-factor were involved in post-translational translocation, we carried out a series of experiments in which major domains were either deleted or fused onto reporter proteins. Fusion of various domains of prepro-alpha-factor onto the reporter protein alpha-globin did not allow post-translational translocation to occur in the yeast in vitro system. Prepro-alpha-factor retained its ability to be post-translationally translocated when parts or all of the pro region were deleted. Removal of the C-terminal repeats containing mature alpha-factor had the most profound influence as post-translational translocation decreased in proportion to the number of repeats deleted. Taken together, these results suggest that efficient post-translational translocation requires a signal sequence and the four C-terminal repeats. There does not however, appear to be specific information contained within the C-terminus, as their presence in fusion did not enable the post-translational translocation of reporter proteins. Lastly, the ability to post-translationally translocate radiochemically pure prepro-alpha-factor that had been isolated by immuno-affinity chromatography required the addition of a yeast lysate fraction. Moreover, post-translational translocation is a function of the microsomal membrane of yeast microsomes and not of a factor peculiar to the yeast lysate, as reticulocyte lysate supported this as well.

Glycosylation↗
PubMed1990

Structural features of the colony-stimulating factor 1 receptor that affect its association with phosphatidylinositol 3-kinase.

The colony-stimulating factor 1 receptor (CSF-1R), immunoprecipitated with either anti-phosphotyrosine or anti-receptor antibodies from lysates of ligand-stimulated cells, is associated with a phosphatidylinositol (PtdIns) 3-kinase activity. The ligand-independent transforming efficiencies of human CSF-1R mutants containing certain amino acid substitutions at codon 301 in their extracellular domains correlated directly with their levels of associated lipid kinase activity. A tyrosine kinase defective CSF-1R mutant (CSF-1R[met616]), containing a mutated ATP binding site, lacked associated PtdIns 3-kinase activity in immune complexes recovered from CSF-1-stimulated cells. However, CSF-1R[met616] associated with PtdIns 3-kinase when phosphorylated in trans in CSF-1-stimulated cells coexpressing an enzymatically competent CSF-1R tyrosine kinase. Another CSF-1R mutant, (CSF-1R[delta KI]), lacking 67 amino acids from its intracellular 'kinase insert' domain, exhibited a partially impaired ligand-dependent mitogenic response and a significant reduction in its associated PtdIns 3-kinase activity. Ligand-stimulated CSF-1R[delta KI] molecules contained levels of phosphotyrosine almost equivalent to wild-type receptors, but were phosphorylated at different sites in vitro. Therefore, the association of CSF-1R with active PtdIns 3-kinase required the receptor tyrosine kinase activity, was triggered by receptor phosphorylation on tyrosine and, in this series of mutants, correlated with their mitogenic potential. Although the receptor KI domain strongly contributes to the association with PtdIns 3-kinase, this region is not strictly essential for the interaction.

Animals↗
PubMed1990

Structural features and adaptive properties of photoreceptors in the skate retina.

In the duplex (rod/cone) retinae of most vertebrates, electrical responses associated with nocturnal vision can be recorded in relative isolation only over a limited intensity range before there is encroachment by signals from the cone system; e.g., at mesopic levels of illumination, the rods begin to saturate, and the cone mechanism is brought into play. On the other hand, the retinae of some sub-species of skate appear to contain only one class of photoreceptors, namely rods. Nevertheless, skate photoreceptors, as well as other retinal neurons, are able to respond to incremental stimuli presented on background fields so intense that 90% or more of the available rhodopsin is bleached during the exposure. These and other findings raise some doubts as to whether the skate has, in fact, an all-rod retina. In this paper, we present a body of evidence--based on the results of photochemical, anatomical, and electrophysiological studies--to support the view that only a single class of photoreceptor subserves vision in the skate retina. In addition, recent findings will be described that: a) demonstrate how the visual cells transform from sluggishly responding rods to brisk, cone-like elements, and b) may provide some insight into the functional significance of this unusual adaptive property.

Adaptation, Ocular↗