Surface versus edge-based determinants of visual recognition.
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Color Perception↗
Biomedical subjects
G Ju
Publications and source records attributed to G Ju.
Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.
Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.
Binding, Competitive↗
[The efferent pathway of the hypothalamus and its functions].
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Animals↗
Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity.
To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.
Amino Acid Sequence↗
Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells.
By use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques. CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 microns to 65 microns. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transection of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI. The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.
Animals↗
Immunohistochemical evidence for a spinothalamic pathway co-containing cholecystokinin- and galanin-like immunoreactivities in the rat.
Using indirect immunofluorescence technique combined with retrograde tracing as well as surgical lesions, a system of spinothalamic neurons containing both galanin- and cholecystokinin-like immunoreactivity has been defined. The cell bodies are located in the lumbar segments L1-L5 with a preferential localization dorsal to the central canal at rostral levels and lateral to the canal at caudal levels. The cells project via the ventral part of the lateral funiculus to the most ventral and posterior parts of thalamus. Here a distinct, varicose terminal network was seen extending caudally from an area lateral to the medial lemniscus, running medially over the medial lemniscus, traversing the parafascicular nucleus and running dorsal to the fasciculus retroflexus into the periventricular gray matter. Transection of various parts of the spinal cord as well as retrograde tracing experiments indicate that the spinothalamic galanin cholecystokinin system represents a crossed pathway. The present results demonstrate that a spinothalamic system can be characterized by its content of galanin- and cholecystokinin-like peptides, two putative messenger molecules. It is only a minor component of the total spinothalamic projection.
Animals↗
Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus.
The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not know to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111-122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G. P. McGregor et al. (1984) Neuroscience 13, 207-216; S. A. S. Shehab and M. E. Atkinson (1984) J. Anat. 139, 725; S. A. S. Shehab and M. E. Atkinson (1986) Expl Brain Res. 62, 422-430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. (Neuroendocrinology 44, 338-346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
c-myc protein and DNA replication: separation of c-myc antibodies from an inhibitor of DNA synthesis.
Antibodies against human c-myc protein have been reported to inhibit DNA polymerase activity and endogenous DNA synthesis in isolated nuclei, suggesting a role for c-myc in DNA replication. Using the same antibody preparations, we observed equivalent inhibition of simian virus 40 DNA replication and DNA polymerase alpha and delta activities in vitro, as well as inhibition of DNA synthesis in isolated nuclei. However, the c-myc antibodies could be completely separated from the DNA synthesis inhibition activity. c-myc antibodies prepared in other laboratories also did not interfere with initiation of simian virus 40 DNA replication, DNA synthesis at replication forks, or DNA polymerase alpha or delta activity. Therefore, the previously reported inhibition of DNA synthesis by some antibody preparations resulted from the presence of an unidentified inhibitor of DNA polymerases alpha and delta and not from the action of c-myc antibodies.
Animals↗
Does cholecystokinin-like immunoreactivity in rat primary sensory neurons represent calcitonin gene-related peptide?
Using immunohistochemistry, calcitonin gene-related peptide (CGRP)- and cholecystokinin (CCK)-like immunoreactivity (LI) were found in many of the same spinal and trigeminal ganglion cells and motoneurons in the spinal cord and hypoglossal nucleus, as well as in fibers with an overlapping distribution in the spinal cord (dorsal horn, bundle ventral to the central canal) and in the spinal trigeminal nucleus. CCK-LI in all these structures disappeared after preadsorption of CCK antisera with CGRP at 10(-4) M and almost completely at 10(-5) M. CCK peptide in concentrations up to 10(-4) M, on the other hand, did not influence CGRP staining. The present findings raise the possibility that some CCK-LI in primary sensory neurons in rat may represent CGRP or a similar peptide.
Animals↗
Projections from the hypothalamus and its adjacent areas to the posterior pituitary in the rat.
Cholera toxin conjugated horseradish peroxidase was injected into the posterior pituitary and its afferents traced in 21 albino rats. The neuronal processes as well as the perikarya were elaborately displayed. The principal and retrochiasmatic supraoptic nuclei and the magnocellular paraventricular subnuclei were densely labelled. The accessory cell groups or nuclei labelled included: the medial preoptic and anterior hypothalamic areas, the anterior and posterior fornical nuclei, the lateral hypothalamic area, the nucleus circularis and nucleus of the forebrain bundle and hitherto unknown or not fully appreciated retrochiasmatic area, the dorsal accessory groups in an area between the stria medullaris and fornix, on the one hand, and the stria terminalis and internal capsule, on the other, and a well developed subependymalperiventricular zone. The medial preoptic nucleus, subfornical organ and organ vasculosum laminae terminalis were also weakly stained. Dendrites of the magnocellular paraventricular nucleus have been said by some to be largely confined to the subnuclei in which they lie. Immunohistochemical studies have proved that they extended beyond their nuclear confinement. The present study has found much wider extension of their dendritic fields. In fact, dendrites of the magnocellular neurosecretory cells in general were long and had a certain degree of directional bias. Several sites projecting to the posterior pituitary were closely related to the cerebrospinal fluid. Namely, the subependymal neuronal plexuses along the third ventricle and beneath the interventricular foramen, and the subpial dendritic plexuses of the supraoptic and retrochiasmatic supraoptic nuclei. Neurons were seen to squeeze in-between the ependymal cells, bringing themselves very close to the cerebrospinal fluid. No direct cerebrospinal fluid-contacting elements, either cell bodies or processes, however, could be ascertained. It is proposed that these plexuses may monitor changes in the cerebrospinal fluid. Besides the principal neurohypophysial tract the posterior pituitary was found in the present study to receive its afferents via two accessory fasciculi, one coursing in the medial forebrain bundle and the other running along the lateral wall of the infundibular recess subependymally.
Afferent Pathways↗
The role of avian retroviral LTRs in the regulation of gene expression and viral replication.
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Alpharetrovirus↗
Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.
mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3. The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.
Base Sequence↗
Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.
A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.
Amino Acid Sequence↗
Transcriptional activity of avian retroviral long terminal repeats directly correlates with enhancer activity.
Retroviral long terminal repeats (LTRs) contain elements responsible for the control of proviral transcription and gene expression. Molecular clones of the LTR region of a number of avian retroviruses have been isolated, and DNA sequence analysis of these clones reveals the existence of a related, but heterogeneous, family of LTRs. To examine the functional significance of the observed sequence differences, we have directly tested the abilities of several different avian retrovirus LTRs to act as promoters and enhancers of mRNA transcription. Our results indicate that large differences in LTR transcriptional activity exist and that these differences in gene expression directly correlate with LTR enhancer activity. In particular, we show that the LTR of Fujinami sarcoma virus is intermediate in both transcriptional and enhancer activity when compared with the very active LTRs of the exogenous viruses RAV-2 and Schmidt-Ruppin B and the much less active LTRs of the endogenous virus RAV-0 and its provirus ev-2. These results suggest that LTR enhancer activity may be the primary determinant of avian retroviral LTR transcriptional activity and, hence, oncogenic potential.
Alpharetrovirus↗
Production of human c-myc protein in insect cells infected with a baculovirus expression vector.
A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.
Animals↗
Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation.
The influence of untranslated 5' sequences on c-myc expression was compared by measuring the translational efficiencies of mRNAs which contain leaders derived from exon 1 or intron 1 of the human c-myc gene. Expression plasmids were constructed and introduced into COS cells, and the levels of c-myc mRNA and protein were examined. Our results show that mRNAs transcribed from constructs containing exon 1 or intron 1, which have different folding potential, are translated with approximately equal efficiencies. This suggests that the translation of c-myc mRNA is not controlled by secondary structure alone. In addition, we observed that transcripts in which exon 1 was deleted are not translated more efficiently, but are present at a higher steady-state level. Thus, this example provides evidence for possible control at the transcriptional level. Finally, since the c-myc product was produced in each of our test systems, the results suggest that this protein does not regulate its own transcription or translation via a specific interaction with c-myc exon 1 alone.
Animals↗
Functional analysis of the transcription control region located within the avian retroviral long terminal repeat.
We used several quantitative assays of in vivo transient gene expression to dissect the elements within the Rous sarcoma virus long terminal repeat (LTR) which constitute the retroviral transcription control region. Site-directed deletion mutagenesis was used to locate and define the enhancer and promoter elements within the LTR. In addition, we inserted exogenous DNA fragments into the LTR to examine the effects of position and sequence on the activity of these LTR transcriptional elements. The Rous sarcoma virus enhancer element, which we propose is located entirely within the LTR, was shown to activate both the beta-globin and retroviral LTR promoters when located in cis. We observed a striking correlation between the degree of activation and the distance between the retroviral promoter and enhancer elements. The LTR promoter element mediated the activation effect of the enhancer element, as LTR deletion mutants containing only the enhancer and TATA box region expressed little activity. The promoter region encoded a low but significant level of transcriptional activity even in the absence of an enhancer. Overall LTR transcriptional activity declined sharply with increasing distance between the LTR promoter and initiator elements. These results shed light on both the importance of the spatial arrangement of the sequence elements within this eucaryotic transcription control region and on the functional interrelationship between these elements.
Animals↗
Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection.
Using a quantitative S1 nuclease protection assay, we demonstrated that acute or chronic infection of avian cells enhances expression of an exogenously introduced rat preproinsulin II gene by approximately equal to 50-fold. The degree of enhancement is shown to vary with the transfection technique used but is independent of the transcription control region of the transfected gene. We conclude that retroviral infection of avian cells enhances expression of transfected DNA in trans by facilitating the uptake of DNA rather than by activating the transfected promoter.
Animals↗