Multiple combinations of alternatively spliced exons in rat tropomyosin-alpha gene mRNA: evidence for 20 new isoforms in adult tissues and cultured cells.

Previous studies of the tropomyosin-alpha gene using Northern blot and ribonuclease protection assay methods identified the expression of nine isoforms generated by alternative splicing of exons. Several of these isoforms were characterized as tissue-specific and/or developmentally specific. The present study used a highly sensitive RT-PCR-based strategy to assay the expression of these and many novel isoforms in a variety of adult rat tissues. All 9 isoforms were found to be expressed in all tissues evaluated. Furthermore, 20 new isoforms were identified with varying tissue specificity. Sequence analysis confirmed exon splicing patterns. This greater degree of isoform generation parallels recent findings for another tropomyosin gene, the TM-5 gene, for which the generation of new isoforms, in particular, ones using novel junctions for carboxy-terminal-coding exons, was also shown. Several of the new cDNA-based isoforms predict tropomyosin protein species that are 10 amino acids longer than previously characterized high-molecular-weight tropomyosin-alpha gene isoforms. The apparent lack of significant tissue specificity in the expression of tropomyosin isoforms suggests that many of these isoforms have more generic roles in cell function.

Insect globin gene polymorphisms: intronic minisatellites and a retroposon interrupting exon 1 of homologous globin genes in Chironomus (Diptera).

Exon 1 of globin gene ct-13RT in clone lambdagb2-1 from Chironomus thummi contains a 444nt SINE (CTRT1). Based on in situ hybridization to polytene salivary gland chromosomes, C. thummi (ct), C. piger (cp) and C. tentans (ctn) contain copies of CTRT1 at multiple chromosomal loci. Genomic PCR amplifications reveal interrupted (ct-13RT) and uninterrupted (ct-13) alleles of the globin gene in the German population of C. thummi maintained in our laboratory, and only uninterrupted alleles or their homologs in different populations of C. thummi, C. piger and C. tentans. PCR amplification did generate different length fragments from cp-13 gene homologs in natural and laboratory C. piger populations that were due to variation in the length of minisatellite expansions of the central introns of the genes rather than a CTRT1-like SINE. Within minisatellite arrays, aligned homologs were more similar than paralogs in a single population, indicating that a tandem cluster of these repeats predated separation of the C. piger populations. The ct-13 genes of several C. thummi populations lack the minisatellites, suggesting their origin in C. piger only after the thummi/piger split. CTRT1 transposition into a ct-13 allele is even more recent, occurring after separation of German and other European C. thummi populations. The nearly intact ct-13RT and comparison with its intact ct-13 allele support a very recent transposition of the CTRT1 SINE into one of at least two already diverged ct-13 globin gene alleles. PCR analysis of DNA from individual adults in C. thummi shows a 1:2:1 distribution of ct-13/ct-13:ct-13/ct-13RT:ct-13RT/ct-13RT genotypes, consistent with a neutral spread of the ct-13RT allele since transposition, and indicating that the hemoglobin encoded by ct-13 is not necessary for survival, at least in a laboratory population of C. thummi.

An electrospray ionization mass spectrometric study of the extracellular hemoglobins from Chironomus thummi thummi.

The aquatic larvae of the dipteran, Chironomus thummi thummi contain extracellular hemoglobins which exhibit stage-specific expression. We have used maximum entropy-based deconvolution of the complex, multiply charged electrospray ionization mass spectra, to demonstrate the presence of more than 20 components, ranging in mass from 14,417.3 Da to 17,356.5 Da in the 4th instar larvae. Of the 15 major peaks with intensities > 10 relative to 100 for the 14,417.3 Da-component (CTT-IV), only the 15,528.2-Da peak does not correspond to a known amino acid sequence. Since the number of C. thummi thummi globin genes now stands at 27, including one cDNA and not counting three that must encode known globins, our results suggest that only a limited number of the globin genes are expressed in the 4th instar larvae.

Evolution of orthologous intronless and intron-bearing globin genes in two insect species.

While globin genes ctt-2beta and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2beta and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species.

Molecular evolutionary analysis of the YWVZ/7B globin gene cluster of the insect Chironomus thummi.

We report the sequence of 8.1 kb of DNA containing the 3' end of one and seven other complete intronless globin genes from the YWVZ/7B locus of the dipteran Chironomus thummi thummi. One of these (ctt-v) appears to be a pseudogene by virtue of a premature termination codon, whereas the others encode apparently functional globin polypeptides. taken together with previously published data, the C. th. thummi YWVZ/7B locus codes for at least 11 globins, five of which differ from one another by no more than two amino acids. In contrast only nine globin genes are found in a comparable genomic clone isolated from C. th. piger. As indicated by sequence alignment, this difference in copy number can be attributed to a loss of one gene (fusion of globin genes 7B8 and 7B10) in the piger lines, coupled with a gain (globin gene 7B9) in the thummi lineage. Comparisons between the thummi and piger sequences showed that YWVZ/7B intergenic regions have maintained a level of 91% similarity since the thummi/piger divergence: most differences are simply due to single base substitutions or insertion/deletion events in either the thummi or the piger DNA, but three instances of partially overlapping deletions were also detected. A phylogenetic analysis of YWVZ/7B gene products was conducted, from which a plausible reconstruction of the evolutionary history of the locus was obtained. In addition, alignment of globin 7B amino acid sequences suggested that globin genes 7B2 and 7B3 (reported at the protein and cDNA level, respectively, but not contained on the C. th. thummi or C. th. piger genomic clones) are possibly chimeric genes. Given the trend toward expansion of the C. thummi globin gene family in general and of the globin 7B subfamily in particular, we propose that increased copy number of these genes has been positively selected as a mechanism to achieve a high Hb concentration in the larval hemolymph.

The dimeric Aalpha chain composition of dysfibrinogenemic molecules with mutations at Aalpha 16.

In the last stage of fibrinogen synthesis, two Aalpha-Bbeta-gamma half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aalpha16 R-->H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA*, FPA*) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aalpha16 R-->H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R-->C (four) or R-->H (three) Aalpha16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot ('N-DSK') cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve 'N-DSK' components with two FPA*'s (N-DSK homodimer), one FPA* (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aalpha chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aalpha chain are produced, both Aalpha chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.

Sequence and evolution of the gene for the monomeric globin I and its linkage to genes coding for dimeric globins in the insect Chironomus thummi.

We isolated genomic clones containing sequences encoding globins I and IA from a Chironomus thummi thummi genomic library. Three clones contain globin IA (ctt-1A) genes, while one contains a globin I (ctt-1) gene. The coding regions of the four genes are identical except for the single base substitution accounting for the globin I/IA polymorphism. The noncoding DNA flanking the coding region is more than 98% similar, confirming a previous hypothesis that the globin ctt-1 and ctt-1A genes are alleles. Hemoglobins I and IA are monomeric in the insect hemolymph. Earlier in situ hybridization studies suggested that monomeric and dimeric globin genes are clustered at different chromosomal loci. In situ hybridization of ctt-1 DNA to polytene salivary gland chromosomes places the ctt-1 gene on the same band as genes for the dimeric globins II beta and VIIB, forcing revision of the earlier hypothesis that genes for monomeric and dimeric globin genes are at different loci. The evolution of the ctt-1 and ctt-1A alleles and of the two globin gene loci are discussed.

Sequence of release of fibrinopeptide A from fibrinogen molecules by thrombin or Atroxin.

During the conversion of fibrinogen to fibrin, two amino-terminal fibrinopeptides A (FPAs) are cleaved by thrombin from each molecule. During early phases of conversion, fibrin intermediates lacking one of two FPAs (des A fibrin) are produced, the level of which depends on whether the FPA cleavage sequence from each molecule is random of concerted. Random cleavage of FPA would produce higher levels of des A fibrin at any thrombin concentration than would concerted cleavage, and the level of this intermediate product would have an important effect on the ultimate structure of the fibrin clot. Because evidence bearing on this subject is conflicting, we carried out experiments to assess the FPA release sequence from fibrinogen by thrombin or by an FPA-cleaving snake venom enzyme, Atroxin. At timed intervals the enzymatic reaction was terminated by precipitation with trichloroacetic acid, and the precipitate was then treated with cyanogen bromide to produce a dimeric amino-terminal fragment. These disulfide-linked amino-terminal fragments of fibrinogen, containing both, one, or neither FPA, were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their distribution quantified by densitometry. The rates of cleavage of the first FPA, k1, and of the second FPA, k2, were computed by fitting the data to equations for a consecutive chemical reaction. This analysis indicated that cleavage by either enzyme resulted in substantial amounts of des A fibrin intermediates. The ratio of the cleavage rates (k2/k1) was higher for thrombin (1.2 +/- 0.3) than it was for Atroxin (0.7 +/- 0.2) but indicates in both cases that the release rate of the second FPA is nearly the same as that of the first FPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequences of globin 6 gene alleles and linkage of globin 6 and 7B genes in the insect Chironomus thummi thummi.

The purpose of this study was to isolate the stage-specifically expressed Chironomus thummi thummi globin-encoding gene (Gb) 6 (ctt-6). The final product of this gene is hemoglobin (Hb) CTT-VI, a protein that is phylogenetically most closely related to Hb CTT-VIIB. In the absence of chromosomal rearrangement, genes of immediate common ancestry should be closely linked. This was shown for a genomic clone containing the Gb ctt-3 and ctt-4 genes (encoding Hb CTT-III and CTT-IV, respectively), and another clone containing the Gb ctt-2 beta and ctt-9a genes (encoding Hb CTT-II beta and CTT-IXa, respectively). We report the isolation and sequence of two alleles of Gb ctt-6 found on independent genomic clones screened with a ctt-6 cDNA, and the predicted linkage of Gb ctt-6 and a ctt-7B gene. The latter, designated Gb ctt-7B9/5, is unusual in being a chimera of two previously reported Gb ctt-7B genes. The result of a partial gene conversion or an unequal crossover between oppositely oriented genes, the chimeric ctt-7B9/5 represents either an additional ctt-7B locus or a 7B haplotype.

Regulation of plasminogen activation by interleukin-6 in human lung fibroblasts.

We determined that exposure of cultured lung fibroblasts (HEL-299) to recombinant human interleukin-6 (0-400 ng/ml) resulted in a dose- and time-dependent increase in secreted and cell lysate PAI-1 and total tPA levels (maximal increase of 2.6-fold and 1.7-fold, respectively). Specificity of this response was indicated when increases in PAI-1 levels were inhibited by neutralizing polyclonal antibodies to IL-6, but not with non-specific antibodies. Inhibition of the response to IL-6 by cycloheximide and alpha-amanitin indicates that increases in PAI-1 are dependent on both protein and RNA synthesis. The addition of IL-6 to HEL-299 cells also stimulated a dose- and time-dependent increase in steady-state PAI-1 mRNA levels (3.8 to 15.1 pg/micrograms total RNA by 24 h). A rapid increase (5-6-fold) in PAI-1 mRNA levels was found between 3 and 12 h. Nuclear run-on assays using a maximum dose of IL-6 showed that IL-6 increases a 4-fold rate of transcription of the PAI-1 gene. We further showed that LPS induces a 70% increase in secreted IL-6 and a 50% increase in PAI-1 protein levels. Increasing doses of anti-IL-6 completely blocked the effect of LPS on PAI-1 while non-specific antibodies had no effect. These studies suggest an autocrine role for IL-6 in regulating localized proteolysis and modulating tissue remodeling during acute inflammatory conditions by fibroblasts.

Intron-containing globin genes in the insect Chironomus thummi.

All previously reported chironomid globin genes are intronless, suggesting that the ancestral chironomid globin gene was also intronless. In this study, the coding regions of the closely linked Chironomus thummi globin (Gbs) II beta and IX genes are shown to be interrupted by noncoding DNA bounded by a 5'-GT and a 3'-AG. Both genes have appropriately placed transcription and translation signals. Polymerase chain reactions on genomic DNA with oligonucleotides flanking and within the putative Gb II beta intron generated products the size predicted for a gene with a 64-nucleotide intron, and sequencing of a cloned PCR fragment also revealed the intron. A partial-length Gb II beta cDNA sequence exactly matches that of the Gb II beta coding regions. We conclude that the intron-containing chironomid globin genes are functional. Regions of the Gb II beta and IX genes spanning the introns are more similar (86%) than the exons themselves (72% similarity), possibly due to partial gene correction. Surprisingly, Gb II beta and IX gene homologues in C. tentans are intronless. If the common ancestor of chironomid globin genes was not intronless, introns were lost in at least three C. thummi globin-gene lineages, and more recently by Gb II beta and Gb IX genes in C. tentans. If, as previously believed, the ancestral chironomid globin gene was intronless, the ancestral chironomid globin gene was intronless, then an intron was recently acquired in only one C. thummi globin sublineage. These alternatives are discussed.

Comparison of the sequence of fibrinopeptide A cleavage from fibrinogen fragment E by thrombin, atroxin, or batroxobin.

In order to investigate the sequence of fibrinopeptide release from the amino terminal end of a dimeric fibrinogen-derived substrate by thrombin or batroxobins, we studied their effects on plasmic fragment E1, a core fragment from the central domain of fibrinogen containing both A alpha chain fibrinopeptide A (FPA) sequences. Isoelectric focussing (IEF) was employed as a means of resolving des A-fragment E1, from which one FPA had been cleaved, from des AA-fragment E1 resulting from the loss of both FPA's. Using densitometric gel scanning for quantification of the levels of intact fragment E1, des A-fragment E1, and des AA-fragment E1, in mixtures incubated with enzyme for various periods of time, we found similar catalytic rate constants (k1, k2) for release of the first fibrinopeptide A, (FPA1) or the second, (FPA2) from fragment E1, with either thrombin or batroxobin (k2:k1 ratios of 1.10 +/- 0.42, 1.34 +/- 0.26 respectively). Atroxin released FPA2 more slowly than FPA1 with a k2:k1 ratio of 0.34 +/- 0.1. Th finding that the cleavage of FPA2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombin cleavage of FPA2 may be cooperative in nature. However, the cooperativity in the cleavage sequence is insufficient to markedly suppress the evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.

Stimulation of chick hepatocyte fibronectin production by fibroblast-conditioned medium is due to interleukin 6.

Interleukin-6 (IL6) is produced by different cell types, including monocytes and fibroblasts. We show that recombinant human IL6 (rhIL6) and chick fibroblast conditioned medium stimulate plasma fibronectin (PFn) and PFn mRNA production by cultured chick hepatocytes in a dose-dependent manner. Lipopolysaccharide (LPS) treatment of fibroblast cultures induces higher levels of the PFn stimulating activity. These effects are blocked by preincubation of either rhIL6 or LPS-stimulated chick fibroblast conditioned medium with anti-rhIL6 antibody before treatment of hepatocytes, indicating that the conditioned medium contains chick fibroblast-derived IL-6 (cfIL6). Further, LPS induces fibroblast production of a proportional increase in cfIL6 detectable by a human IL6 ELISA. cfIL6 maximally stimulates chick hepatocyte PFn production by 24 h (4.5-fold). Dexamethasone acts more rapidly, but maximal stimulation is only 2.3-fold. Hepatocyte Fn mRNA levels are even more substantially stimulated by dexamethasone and cfIL6 (up to 8.9- and 18.5-fold by 12 h, declining to 2.3 and 4.2-fold by 24 h, respectively). The effect cfIL6 with or without dexamethasone on hepatocyte PFn levels are comparable. These observations are consistent with the role of IL6 as a major mediator of acute phase protein production.

The cleavage sequence of fibrinopeptide A from fibrinogen fragment E by thrombin, atroxin or batroxobin.

Calculations of data from fibrin polymerization and cross-linking experiments infer that thrombin-catalysed release of the second of the two fibrinopeptides A (FpA2) from fibrinogen is concerted, although other data suggest that FpA2 release is random. In the concerted pattern of FpA release, divalent monomer (des AA-fibrin) formation predominates throughout the enzymatic conversion of fibrinogen to fibrin, an effect leading to relatively rapid fibril assembly. Alternatively, random FpA2 release would result in a substantial population of monovalent monomer (des A-fibrin) intermediates during early and intermediate phases of the enzymatic conversion to fibrin. Their formation would cause a delay in fibrin fibril assembly. In order to address the question of the pattern of FpA release directly, we purified plasmic fibrinogen fragment E1 isoforms containing both FpA sequences and studied the sequence of FpA release by thrombin or batroxobin. Des A-fragment E1 intermediates formed by loss of one FpA (FpA1), and des AA-fragment E1 products (lacking both FpA1 and FpA2) were identified by analytical isoelectric focusing and quantified by densitometry. The catalytic rate of release of FpA1 (k1) and FpA2 (k2) by thrombin or batroxobin was similar. The ratio of these rates, k2:k1, was 1.10 +/- 0.42 for thrombin and 1.34 +/- 0.26 for batroxobin. These findings indicate that these enzymes cleave FpA2 randomly from fragment E1.

Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.

We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels.

Differential regulation of insect globin and actin mRNAs during larval development in Chironomus thummi.

S1 nuclease protection assays were used to measure changes in the steady-state levels of six different globin (Gb) mRNAs in the midge, Chironomus thummi thummi (C. thummi, Diptera) during larval development. Two distinct patterns of change were observed. GbI, IV, VIIB-4 and VIIB-5 transcripts were present in 3rd instar larvae, rose from low levels immediately post-moult to peak levels by day 2-3 of the 4th instar, and then declined, reaching near-basal levels by day 7-8. In contrast, transcripts of GbIII (known from previous studies to be 4th instar-specific) and VI, which were undetectable in the 3rd instar, rose to high levels by day 2 of the 4th instar, but remained elevated thereafter. Our data further showed that closely linked Gb genes were not necessarily expressed in a coordinate manner. Unlike the Gb mRNAs, actin (Act) mRNA levels (measured by slot-blot hybridization to a heterologous probe) increased progressively as a proportion of total RNA during 4th instar development. Therefore, the regulation of C. thummi Gb transcript levels is specific, differing from that of Act and among the Gb mRNAs themselves. Elevated 20-hydroxyecdysone (HE) titer at the 3rd-4th instar moult correlates with the low steady-state levels of Gb mRNAs immediately post-moult. However, other aspects of Gb mRNA profiles cannot be explained on the basis of a direct repressive effect by HE on Gb gene transcription.

Multiple clustered genes of the haemoglobin VIIB subfamily of Chironomus thummi thummi (Diptera).

The nucleotide and inferred amino acid sequence of three globin VIIB genes from the midge, Chironomus thummi thummi have been determined. These three genes are intronless, like all previously reported Chironomid globin genes. Transcription of globin genes VIIB-4, VIIB-5 (and/or VIIB-6) and VIIB-7 was demonstrated by S1 nuclease protection analysis. Comparison of actual and inferred globin VIIB amino acid sequences reveals that (i) most of the amino acid substitutions are conservative in nature, and (ii) none of the substitutions are expected to interfere with the ability of the globins to bind haem. Nucleotide sequence comparisons suggest that gene duplications and partial gene corrections (conversions) have occurred recently in the globin VIIB subfamily locus.

Regulation of plasma fibronectin biosynthesis by glucocorticoids in chick hepatocyte cultures.

Plasma fibronectin is an acute-phase reactant synthesized by hepatocytes. Glucocorticoids are one of the major factors implicated in controlling the hepatic acute-phase response. To study the regulatory effects of glucocorticoids on plasma fibronectin biosynthesis, a model chick hepatocyte culture system under serum- and hormone-free conditions was used. In the presence of either dexamethasone or corticosterone, secreted plasma fibronectin increased maximally to 2.8-fold above basal levels. The stimulatory effect of the hormones was maintained only in their continuous presence, since plasma fibronectin production dropped to near basal levels within 16 h of glucocorticoid withdrawal. Pulse-chase studies indicated that pretreatment of cells with dexamethasone stimulated the level of secreted plasma fibronectin but had no effect on its rate of secretion. The increase in plasma fibronectin production by dexamethasone was abolished in a dose-dependent manner by the addition of progestin, an antagonist of dexamethasone known to compete specifically for the liver glucocorticoid receptor. Actinomycin D and alpha-amanitin, which are inhibitors of transcription, also blocked the early dexamethasone effect on plasma fibronectin synthesis. Slot blot hybridization of total RNA samples from dexamethasone-treated cultures revealed a 6-fold stimulatory rise in fibronectin mRNA during the first 6 h of treatment, which later declined and was no longer evident at 48 and 72 h. However, fibronectin mRNA levels were elevated to about the same extent in control and dexamethasone-treated cells at the later time points. During the same time period (0 to 72 h), plasma fibronectin protein levels rose and remained elevated. Evaluation of pulse-chase experiments following pretreatment with hormone for 48 h demonstrated that equal amounts of plasma fibronectin were translated by dexamethasone-treated and control cells, but only 42% of labeled plasma fibronectin was secreted by control cells compared with 93% for dexamethasone-treated cells. These findings suggest that the early phase of glucocorticoid regulation of plasma fibronectin biosynthesis occurs at the transcriptional level and is mediated through the specific action of the glucocorticoid receptor. A later phase of glucocorticoid-stimulated plasma fibronectin biosynthesis results from modulation of post-translational processing events leading to secretion of an increased amount of newly translated plasma fibronectin polypeptides.

Post-embedding direct immunogold detection of a protein antigen in insect tissue.

An immunocytochemical study was performed to localize the site of hemoglobin synthesis in larvae and embryos of the insect Chironomus thummi. Heterologous antisera specific for C. thummi hemoglobins were prepared using a highly purified hemoglobin extract. Tissue samples were prepared by glutaraldehyde fixation of whole dissected larvae or whole embryos without osmium tetroxide postfixation. Epoxy resin-embedded thin sections were labeled with a direct immunogold conjugate. Immune label was localized in rough endoplasmic reticulum of fat body cells of larvae. Immune label was also present in embryos. The technique, which did not require chemical etching of the sections, proved very useful for demonstration of this intracellular protein antigen.