Identification and characterisation of the egr-1 gene product as an inducible, short-lived, nuclear phosphoprotein.

The egr-1 gene (also termed NGFI-A, zif/268 and Krox24) is predicted to encode a protein with three 'zinc finger' domains. Analogous DNA-binding motifs are present in several putative transcriptional regulatory proteins. The mRNA of egr-1 is rapidly and transiently induced by a variety of growth factors in diverse cell types. We have used a synthetic peptide corresponding to the C-terminus of the predicted egr-1 protein to generate mouse and rabbit polyclonal antibodies. These antibodies have been used to identify the egr-1 protein, designated p75egr-1, by immunoprecipitation, immunoblotting and immunocytochemical analyses. p75egr-1 is induced within 1-2 h of mitogenic stimulation and has a half-life of under 2 h. The protein is phosphorylated and has been localised to the nucleus by both immunocytochemical analysis and biochemical fractionation. The egr-1 protein is released from nuclei under isotonic or hypertonic salt conditions or by treatment with DNAase and binds double-stranded DNA in vitro. Consistent with the distribution described for the mRNA the egr-1 protein is abundantly expressed in normal rat brain.

c-myc protein expression in untransformed fibroblasts.

We have examined and quantitated the expression of c-myc protein in two untransformed fibroblast cell lines, murine Swiss 3T3 and human MRC-5, c-myc protein is not detectable in quiescent cells, but it is rapidly induced upon mitogenic stimulation. Peak expression is seen about 3-5 h after serum stimulation, and corresponds to about 3-6000 molecules per cell (mpc). Thereafter, levels fall back to a quiescent level in confluent fibroblasts, but remain elevated at 1-3000 mpc in subconfluent cells. The c-myc protein is phosphorylated and has the same size and short half-life as seen in tumour cells. Removal of serum growth factors from the culture medium causes very rapid loss of the c-myc protein from all cells, irrespective of their positions in the cell cycle. Thus, c-myc expression is continuously dependent upon the presence of mitogens. However, no single tested mitogen is obligatory for maintenance of expression in proliferating cells. Growth arrest of cells, either by metabolite starvation or by drugs which inhibit DNA synthesis, does not affect expression of the c-myc protein, which remains completely dependent upon the presence of mitogens. These data are consistent with the c-myc protein's having a continuous role in proliferating cells as an intracellular integrator of growth regulatory signalling pathways.

A sensitive enzyme-linked immunosorbence assay for the c-fos and v-fos oncoproteins.

The c-fos nuclear oncoprotein is rapidly induced when the growth of normal cells is initiated by mitogens, and it is also synthesized in several cell systems in response to stimuli that do not cause cell proliferation. When expressed inappropriately, c-fos, and its retroviral counterpart v-fos, can transform susceptible cells in vivo and in vitro. We have developed a simple and sensitive ELISA for the c-fos and v-fos proteins. Fos proteins are captured from cell lysates by an antibody specific for an amino-terminal peptide substantially conserved between v-fos and c-fos; the captured proteins are recognised by a second antibody against a different peptide sequence also conserved in the two proteins. The second antibody has been conjugated to alkaline phosphatase to provide an enzyme label; bound alkaline phosphatase is measured with a sensitive cycling enzyme system that generates a coloured end-product. We show that the fos ELISA is immunologically specific and use it to monitor increased c-fos expression in serum-stimulated HeLa cells and human fibroblasts, and in mitogen-stimulated murine thymocytes.

Adenine phosphoribosyltransferase deficiency with renal deposition of 2,8-dihydroxyadenine leading to nephrolithiasis and chronic renal failure.

Homozygous adenine phosphoribosyltransferase deficiency is a genetic defect that is associated with 2,8-dihydroxyadenine urolithiasis. Since the prevalence of the heterozygous state is found in 0.4% to 1.2% of the population, it is surprising that more cases of 2,8-dihydroxyadenine urolithiasis have not been reported. Herein we describe a patient with complete adenine phosphoribosyltransferase deficiency with 2,8-dihydroxyadenine urolithiasis leading to chronic renal failure. Gene sequencing revealed that the patient is a compound heterozygote. One of the mutations (a T insertion between bases 346 and 347) has been encountered before, but the second (a G-to-A substitution at base 1356) has not been previously reported. Possible explanations for the unexpected rarity of 2,8-dihydroxyadenine urolithiasis are discussed.

Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor in unstressed or stressed male rats.

Corticotropin-releasing factor has an integrative role on the behavioral, endocrine and autonomic responses to stress. Immediate-early gene (c-fos) expression was used to determine patterns of neural activity in the limbic system following i.c.v. infusion of corticotropin-releasing factor. Either 250 or 1000 pmol corticotropin-releasing factor infused into the lateral ventricle of precannulated and handled male rats resulted in marked c-fos expression 60 or 120 min later in localized regions of the basal forebrain, including the ventrolateral septum, the dorsal and medial parvicellular divisions of the paraventricular nucleus, the central nucleus of the amygdala, and dorsal bed nucleus of the stria terminalis. Pre-infusion of alpha-helical corticotropin-releasing factor (2500 pmol), a competitive corticotropin-releasing factor antagonist of corticotropin-releasing factor, had no effect on immediate-early gene expression alone but reduced that elicited by exogenous i.c.v. corticotropin-releasing factor (250 pmol)--in some areas to control levels. Fifteen minutes of restraint stress, a situation in which corticotropin-releasing factor is released endogenously, also activated c-fos expression in a pattern that resembled corticotropin-releasing factor infusions but was not identical. There was enhanced expression in the dorsal and medial areas of the paraventricular nucleus, but not its magnocellular region, and increased expression in the ventrolateral septum; however, there was no detectable response on the central amygdala. Preinfusion of alpha-helical corticotropin-releasing factor (2500 pmol) had no significant effect on stress-induced c-fos expression in the ventrolateral septum or paraventricular nucleus. This suggests that corticotropin-releasing factor release may form only a part of the central neurochemical response to restraint stress. Rats given i.c.v. corticotropin-releasing factor (250 pmol) before restraint stress showed additive effects on c-fos in the ventrolateral septum but not in the paraventricular nucleus; the central nucleus of the amygdala reacted as if corticotropin-releasing factor alone had been infused. Corticosterone levels were raised by both stress and corticotropin-releasing factor, but pretreatment with alpha-helical corticotropin-releasing factor reduced them after either procedure, which correlates with c-fos expression in the paraventricular nucleus and ventrolateral septum. These results show that corticotropin-releasing factor induces a specific pattern of c-fos expression in localized regions of the amygdala, hypothalamus and septum, which may indicate a corresponding pattern of neural activation. Restraint, one form of stress, activates c-fos in a similar but not identical manner, suggesting that corticotropin-releasing factor may not be the only neuropeptide involved in the response to this stressor.(ABSTRACT TRUNCATED AT 400 WORDS)

Renal insufficiency secondary to 2,8-dihydroxyadenine urolithiasis.

A 48-year-old man with a history of recurrent urolithiasis and chronic renal failure underwent a nephrectomy for a renal mass. At surgery the mass proved to be a calculus impacted in a dilated calyx. Gross examination of the kidney revealed chalky white deposits in the deep medulla and papillary tips. Histologic examination revealed chronic interstitial nephritis with brown spicules within some tubular epithelial cells and larger deposits of brown crystals within tubular lumina, the interstitium of the medulla, and papillary tips. Polarization microscopy revealed individual crystals scattered throughout the renal parenchyma. Although the arrangement of the crystals was reminiscent of uric acid, and, in fact, a clinical diagnosis of gouty nephropathy was made, x-ray diffraction analysis demonstrated crystals of 2,8-dihydroxyadenine. Enzymatic studies confirmed the complete absence of adenine phosphoribosyltransferase activity in erythrocyte lysates.

The role of N-methyl-d-aspartate-type glutamatergic neurotransmission in the photic induction of immediate-early gene expression in the suprachiasmatic nuclei of the Syrian hamster.

Abstract This study investigated the role of N-methyl-D-aspartate (NMDA)-type glutamatergic neurotransmission in mediating the photic induction of immediate-early gene expression in the Suprachiasmatic nucleus (SCN) of the Syrian hamster. Activation of c-fos, c-jun and egr-1 was assessed by immunocytochemical detection of their protein products. To characterize the circadian basis to the inductive effects of light, hamsters were allowed to free-run in constant dim red light and received a 1 h light pulse at different circadian phases relative to activity onset (defined as CT 12). In control animals which did not receive light pulses, c-fos and egr-1 expression was absent or restricted to a small area of the dorsolateral region of the SCN, and expression of c-jun could not be detected in the SCN. In hamsters killed after presentation of a light pulse at either CT 14 or CT 20, there was a marked increase in c-fos and egr-1 immunoreactivities throughout the ventrolateral division of the SCN. In contrast, light pulses given at CT4 or CT 8 failed to activate immediate-early gene expression. Light pulses did not induce c-jun immunoreactivity at any circadian phase tested. Staining for c-fos was maximal 1 h after the start of the light pulse and had started to decline by 2 h. At this later time, c-jun expression was still undetectable. To compare the distribution of retinal afferents with that of c-fos induction, hamsters held on a light schedule of 16 h light: 8 h dark received an intraocular injection of cholera toxin-horseradish peroxidase conjugate 3 days before exposure to a 1 h light pulse given 2 h after lights off. Comparison of adjacent sections processed for c-fos immunoreactivity or for cholera toxin-horseradish peroxidase revealed that light-induced c-fos expression was precisely restricted to retinal terminal fields in the SCN. Light pulses also induced c-fos expression in the retinoreceptive ventral lateral geniculate nucleus and intergeniculate leaflet but not in the retinal fields of the dorsal lateral geniculate nucleus, indicating that the expression of cfos in response to light is spatially specific. The aim of the subsequent experiments was to investigate the role of NMDA-type glutamatergic neurotransmission in mediating the effects of light on c-fos expression in the SCN. To determine whether NMDA had the potential to activate c-fos expression in the SCN, hamsters were infused with 2.5 nmol NMDA or vehicle via an intracerebroventricular (icv) cannula positioned adjacent to the nuclei. In contrast to the effects of light, icv NMDA activated c-fos expression at both CT8 and CT 14. The distribution of immunoreactivity was more widespread than that observed after light, extending throughout the SCN and adjacent hypothalamus. To test whether NMDA receptors had a physiological role in the photic response, hamsters were treated systemically with the non-competitive NMDA antagonist MK801 (dose range 0.6 to 6.0 mg/kg body wt, ip) or vehicle prior to exposure to a 1 h light pulse given at CT 14 or CT 20. Expression of c-fos was still detectable in the dorsolateral SCN but MK801 blocked expression in the ventral portion of the retinoreceptive zone of the SCN. MK801 (10 or 100 nmol) delivered centrally (icv) also prevented light-induced c-fos expression in the ventral region of the SCN bordering the optic chiasm, though staining again persisted in the dorsolateral region. The induction of c-fos by icv NMDA, and the partial blockade of light-induced c-fos expression by the antagonist MK801, are consistent with the hypothesis that glutamate mediates the effects of light on SCN activity. However, the persistent photic induction of c-fos expression in a subfield of retinal afferents following treatment with MK801 suggests that other, non-NMDA-type mechanisms may contribute to photic entrainment.

Synthetic peptides in biochemical research.

Synthetic peptides play an important role in many areas of biological research. Advances in synthetic chemistry and automation over the past few years have resulted in increasingly reliable and rapid syntheses. As a result, peptides are now frequently employed in immunological studies, structural studies, as enzyme substrates, in ligand/receptor studies, and as probes for a range of molecular interactions. This review describes solid-phase peptide synthesis and the applications of synthetic peptides in molecular biology and biochemistry.

FosB in the suprachiasmatic nucleus of the Syrian and Siberian hamster.

The suprachiasmatic nucleus (SCN) generates circadian rhythms of behavior and hormone secretion in mammals, and integrates responses to light and nonphotic stimuli to synchronize such rhythms with the external environment. Previous studies have demonstrated a close association between the induction of the immediate early gene (IEG) c-fos in the SCN by light and phase shifts of circadian rhythms induced by light, but nonphotic stimuli (e.g., arousal), which also cause phase shifts, do not increase c-fos expression in the SCN. Because c-fos is now known to be a member of a large family of IEGs which can regulate transcription and thus cellular function, the aim of the current study was to determine whether induction of another member of this immediate early gene family, fosB, is associated with photic and nonphotic phase shifts. An antiserum that recognizes a unique peptide sequence derived from FosB was produced so that the expression of fosB could be investigated in cells within the SCN by immunocytochemical detection of its protein product. The regional distribution of FosB-immunoreactive (ir) cells in the SCN of Syrian and Siberian hamsters was broadly similar to that for c-Fos-ir cells. However, whereas c-fos expression in the SCN was constitutively low, but could be massively induced by light at particular circadian phases, FosB-ir cells were present at all circadian phases studied, irrespective of photic stimulation, and light only produced marginal increases in the number of FosB-ir cells compared with nonstimulated controls. Moreover, blockade of glutamatergic neurotransmission by pretreatment of hamsters with the NMDA receptor antagonist MK801 significantly reduced photic induction of c-Fos-ir cells, but did not influence the number of FosB-ir cells in the SCN. Finally, an arousing nonphotic stimulus known to cause phase advances in wheel-running behavior in Syrian hamsters did not alter significantly the number of FosB-ir cells in the SCN. These observations indicate that light and nonphotic stimuli are not potent regulators of fosB expression in the SCN. However, because fosB and c-fos can be present in the SCN at the same time after a light pulse, these studies indicate the potential for interactions with each other and with members of the Jun family in the regulation of the circadian timing system.

Synthesis of peptides for use as immunogens.

An increasing problem in cell and molecular biology is the preparation of antibodies specific to proteins that are present in minute quantities within cells or tissues. With the advent of recombinant DNA technology, it is now often possible to deduce the primary amino acid sequence of a polypeptide without its purification. Two strategies then exist to raise appropriate antibodies. Either the gene can be expressed in a heterologous species, usually bacteria, and the resultant protein used as an immunogen, or alternatively, small synthetic peptides can be made that contain amino acid sequences inferred from that of the gene. Such antipeptide antibodies crossreact with the intact native protein with surprisingly high frequency and have the additional advantage that the epitope recognized by the antibody is already well defined (1). In this way, antibodies can be raised against novel gene products that are specifically directed against sites of interest, for example, unique regions, highly conserved regions, active sites, extracellular or intracellular domains. Moreover, the ready availability of the pure peptide immunogen against which the antibody was raised means that sera can be rapidly and easily screened, e.g., using an enzyme-linked immunosorbent assay (ELISA) for antipeptide activity. Free peptide can also be used to block antibody binding and so demonstrate immunological specificity, and it may be coupled to a solid support (e.g., agarose) to generate an affinity matrix for antibody purification.

Production and characterization of antibodies against synthetic peptides.

Immunization protocols vary greatly between laboratories. In general, there are no hard and fast rules and most protocols give satisfactory results. The methods described below are designed to give optimal results with minimal injury to the test animal, and we have used them extensively and successfully for several years (1-5). Peptide immunizations differ from those in which the immunogen is a larger macromolecule in that maximal antipeptide titers (which arise rapidly after 2-3 immunizations) do not always coincide with maximal titers against the intact protein (which tend to peak rather later at 4-6 immunizations). Thus, although antipeptide enzyme-linked immunosorbent assays (ELISAs) are useful gages of immune response, there is no substitute for eventual screening on the intact protein (e.g., by immunoprecipitation, Western blotting, and so on). Individual variation in antipeptide response is very marked, so it is advisable to use several animals (three to six) per immunogen. Rabbit responses are generally poorer in specific pathogen-free (SPF) animals, probably reflecting their greater immune naivity. Mouse responses are often best in F(1) crosses (e.g., BALB/c × C57B1/6) rather than pure strains. Alternatively, SJL mice generally respond well.

Production and characterization of antibodies against synthetic peptides.

Immunization protocols vary greatly between laboratories. In general, there are no hard and fast rules and most protocols give satisfactory results. The methods described below are designed to give optimal results with minimal injury to the test animal, and we have used them extensively and successfully for several years (1-5). Peptide immunizations differ from those in which the immunogen is a larger macromolecule in that maximal antipeptide titers (which arise rapidly after 2-3 immunizations) do not always coincide with maximal titers against the intact protein (which tend to peak rather later at 4-6 immunizations). Thus, although antipeptide enzyme-linked immunosorbent assays (ELISAs) are useful gages of immune response, there is no substitute for eventual screening on the intact protein (e.g., by immunoprecipitation, Western blotting, and so on). Individual variation in antipeptide response is very marked, so it is advisable to use several animals (three to six) per immunogen. Rabbit responses are generally poorer in specific pathogen-free (SPF) animals, probably reflecting their greater immune naivity. Mouse responses are often best in F(1) crosses (e.g., BALB/c × C57B1/6) rather than pure strains. Alternatively, SJL mice generally respond well.

Synthesis of peptides for use as immunogens.

An increasing problem in cell and molecular biology is the preparation of antibodies specific to proteins that are present in minute quantities within cells or tissues. With the advent of recombinant DNA technology, it is now often possible to deduce the primary amino acid sequence of a polypeptide without its purification. Two strategies then exist to raise appropriate antibodies. Either the gene can be expressed in a heterologous species, usually bacteria, and the resultant protein used as an immunogen, or alternatively, small synthetic peptides can be made that contain amino acid sequences inferred from that of the gene. Such antipeptide antibodies crossreact with the intact native protein with surprisingly high frequency and have the additional advantage that the epitope recognized by the antibody is already well defined (1). In this way, antibodies can be raised against novel gene products that are specifically directed against sites of interest, for example, unique regions, highly conserved regions, active sites, extracellular or intracellular domains. Moreover, the ready availability of the pure peptide immunogen against which the antibody was raised means that sera can be rapidly and easily screened, e.g., using an enzyme-linked immunosorbent assay (ELISA) for antipeptide activity. Free peptide can also be used to block antibody binding and so demonstrate immunological specificity, and it may be coupled to a solid support (e.g., agarose) to generate an affinity matrix for antibody purification.

Studies on the interaction of the human c-myc protein with cell nuclei: p62c-myc as a member of a discrete subset of nuclear proteins.

We have analyzed the localization of the human c-myc product (p62c-myc) at steady state in cells by immunoprecipitation and immunoblotting. We show that p62c-myc is extracted from nuclei by mild salt concentrations (below 200 mM), without affecting gross nuclear structure or causing extraction of major chromatin components. We observe no association between p62c-myc and the nuclear matrix. We also demonstrate that p62c-myc is a member of a discrete subset of nuclear proteins that are all rendered irreversibly insoluble in situ by exposure of isolated nuclei to physiological temperatures (37 degrees C). p62c-myc is sequestered into a similar insoluble complex in cells that have been subjected to heat shock. Finally, we show that avian v-myc and v-myb proteins in isolated nuclei also become insoluble after exposure to temperatures above 37 degrees C. We discuss the possible implications of these results.

Intestinal barrier to large particulates in mice.

Intestinal barrier function in mice was assessed after acute or chronic oral administration of 15.8- and 5.7-micron synthetic spherical particles. The results failed to confirm previous reports that ingested particles rapidly appear in blood. Furthermore, 15.8-micron particles did not accumulate in intestinal Peyer's patches, mesenteric lymph nodes, or other organs of the reticuloendothelial system, even after the maximum dosage of 8 X 10(6) particles per day for 60 d. However, the 5.7-micron particles were demonstrated in Peyer's patches, mesenteric lymph nodes, and lungs after the maximum dosage of 4.5 X 10(8) particles per day for 60 d. At 77 d after the termination of ingestion, 5.7-micron particles were still present in these tissues. The 5.7-micron particles were not found in spleen; retention in liver was equivocal. The site of uptake of particles capable of penetrating the intestinal mucosa appears to be the Peyer's patches. It is suggested that most absorbed particles are sequestered in Peyer's patch macrophages. Particles that escape sequestration are transported by lymph rather than by portal blood. The findings indicate that hazards associated with intestinal uptake of large (> 5 micron) particulates exist, but that the frequency of such penetration is still unclear.

Characterization of a heat shock-induced insoluble complex in the nuclei of cells.

The formation of an insoluble complex in isolated nuclei incubated at physiological temperature (37 degrees C) is demonstrated. A similar complex is shown to form in the nuclei of intact cells subjected to temperatures that induce the classical heat-shock response. The formation of this complex occurs rapidly in response to hyperthermia and is induced by small increases in temperature both in vitro and in vivo. We have characterized the formation of the complex in isolated nuclei and the nuclei of intact cells. A small number of the subset of nuclear proteins involved in the complex have been identified. The significance of the loss of solubility of these proteins in the nucleus following hyperthermia is discussed.