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C Abate

Publications and source records attributed to C Abate.

At least 19 recordsLinked to original sources
PubMed1996

Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l-1 h-1. No levan was produced even if a sucrose-based medium was used.

Ethanol↗
PubMed1995

Multiple amino acids determine the DNA binding specificity of the Msx-1 homeodomain.

This study investigates the sequence features that contribute to the differential DNA binding properties of two divergent homeodomains, Msx-1 and HoxA3. We show that these homeodomains have overlapping, but nonidentical, DNA binding site preferences. We defined the amino acid residues that contribute to the observed differences in DNA binding specificity by producing a series of mutated polypeptides in which selected residues in Msx-1 were replaced with the corresponding ones in HoxA3. These analyses show that the DNA binding specificity of Msx-1 versus HoxA3 results from the cumulative action of multiple residues in all segments of the homeodomain (i.e., the N-terminal arm and helices I, II, and III). Therefore, substitutions of residues in both helix III and the N-terminal arm (but not in either segment alone) produced an Msx-1 polypeptide whose binding site preference was indistinguishable from that of HoxA3. Residues in helices I and II also influence DNA binding activity; these oppositely charged residues (e.g., lysine 19 and glutamate 30) may mediate ionic interactions between helices I and II which stabilize DNA binding by Msx-1. These findings demonstrate a critical interplay between residues in each homeodomain segment for appropriate conformation of the protein-DNA complex.

Amino Acid Sequence↗
PubMed1995

Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

3T3 Cells↗
PubMed1994

Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.

Alanine↗
PubMed1994

DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.

We have incorporated the DNA-cleaving moiety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthroline-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA.

Animals↗
PubMed1994

Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster.

The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

3T3 Cells↗
PubMed1993

Dimerization and DNA binding alter phosphorylation of Fos and Jun.

Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcriptional factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here we show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34cdc2 (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation.

Animals↗
PubMed1993

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

Amino Acid Sequence↗
PubMed1992

An evaluation of several laboratory tests and test combinations in the detection of lupus anticoagulant.

The laboratory assessment of the lupus anticoagulant, a factor frequently associated with venous and arterial thrombosis, recurrent miscarriages and abortions, is not straightforward, as indicated by the variety of tests proposed and the different results obtained. On account of the marked variability and heterogeneity of lupus anticoagulant among patients, no single test or reagent will identify all patients with lupus anticoagulant, and a panel of several tests has to be used. This is time consuming and increases the workload of the laboratory. The aim of this study was to assess the minimum number of tests necessary for the satisfactory identification of the patient with lupus anticoagulant. Our study confirms that lupus anticoagulant may be present in a significant number of patients with normal routine activated partial thromboplastin time, a test which therefore cannot be used as the sole criterion for identifying patients suspected of having lupus anticoagulant. In contrast all patients who had positive results in at least one test could be detected (100% sensitivity) with two combinations of tests: (1) dilute activated partial thromboplastin time and Kaolin clotting time and (2) dilute activated partial thromboplastin time and tissue thromboplastin inhibition test. Since the latter inhibition test has been reported to give a high number of false-positive or negative results, we suggest the combination of dilute activated partial thromboplastin time and Kaolin clotting time as the standard pair of tests for the screening of suspected lupus anticoagulant patients.

Antiphospholipid Syndrome↗
PubMed1992

Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

Blood↗
PubMed1991

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.

Amino Acid Sequence↗
PubMed1991

Species-specific distribution of aromatic L-amino acid decarboxylase in the rodent adrenal gland, cerebellum, and olfactory bulb.

Aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-dopa to dopamine, displayed species-specific differences in both activity and immunoreactivity in the cerebellum, olfactory bulb, and adrenal glands of three rodent species, the hamster, rat, and mouse. Specifically, in the hamster but not the rat or mouse, AADC immunoreactive cells were observed in the cerebellum and adrenal cortex. The unusual distribution of the enzyme was confirmed biochemically. AADC activity was greater in the adrenal gland and the cerebellum in the hamster than in the mouse or rat. In addition, by Western blot analysis, one band of appropriate molecular weight was observed both in the hamster adrenal gland and cerebellum. The rat adrenal gland displayed a similar immunoreactive protein on the Western blot; however, the protein could not be detected in the rat cerebellum by the technique utilized. Tyrosine hydroxylase (TH) immunoreactivity in these same tissues did not differ among the species. In the main olfactory bulb of the mouse, juxtaglomerular cells exhibited very limited immunoreactivity for AADC, but TH-immunoreactivity in these cells was robust. In contrast, juxtaglomerular cells in the rat displayed a similar intensity of immunostaining for both AADC and TH. AADC activity in the mouse, consistent with the reduced immunostaining for the enzyme, was 50% of that in the rat and the hamster. These data demonstrate that AADC protein, which is contained in cells of diverse function, also displays qualitative and quantitative species specific variations in both distribution and amount.

Adrenal Glands↗
PubMed1991

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains.

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

Binding Sites↗
PubMed1991

Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity.

Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.

Animals↗
PubMed1991

Fos is phosphorylated by p34cdc2, cAMP-dependent protein kinase and protein kinase C at multiple sites clustered within regulatory regions.

The proto-oncogene c-fos encodes a nuclear protein (Fos) that functions in transcriptional regulation in response to extracellular signals. Fos is extensively modified in the nucleus by serine and threonine phosphorylation. It has been suggested that phosphorylation may play an important role in regulating Fos function in normal and transformed cells. As a first step in addressing this issue, we have used purified Fos as a substrate for several serine-threonine protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and p34cdc2. Each of these kinases phosphorylated Fos at several unique sites. These sites were located within two regions that were previously shown to reduce the transcriptional activity of Fos in vitro. Several of the sites modified in vitro were also shown to be phosphorylated in serum-stimulated fibroblasts. These findings demonstrate that Fos is a target for several protein kinases involved in signal transduction and suggest that phosphorylation could regulate the transcriptional properties of Fos.

Amino Acid Sequence↗