Design and synthesis of a new bicyclic dipeptide isostere.
The synthesis of a new Gly-Pro turn mimetic and the computational study of its ability to induce beta-turn is reported.
Computer Simulation↗
Biomedical subjects
A Brandi
Publications and source records attributed to A Brandi.
Total synthesis of (-)-rosmarinecine by intramolecular cycloaddition of (S)-malic acid derived pyrroline N-oxide.
[reaction in text] Straightforward total syntheses of (-)-rosmarinecine have been achieved from L-malic acid derived pyrroline N-oxides by two novel useful cascade processes, which join the family of domino reactions. Both strategies, which furnished the target alkaloid in enantioenriched and enantiopure forms, respectively, allow complete control of configuration at all the three newly created contiguous stereogenic centers.
Alkaloids↗
Stereoselective approach to hydroxyindolizidines: protection/deprotection of the nitrone functionality via cycloaddition/retrocycloaddition.
The enantiomerically pure indolizidine (-)-21 has been synthesized starting from L-malic acid. The key intermediate 20 has been assembled through an intramolecular 1,3-dipolar cycloaddition of a nitrone generated in situ by retrocycloaddition from isoxazolidine 17 or 18. The configuration of the new three stereocenters was set up with complete control in the cycloaddition step. The presented synthetic route provides a general and highly selective methodology toward indolizidines having the [1,8a]-cis configuration.
Hydroxylation↗
Regio- and stereoselective cycloadditions of cyclic nitrones to maleic diamide forced in a peptide: synthesis of potent ligands of human NK-2 receptor.
The regioselectivity and the stereoselectivity induced by relatively small peptidomimetic maleic diamide 1 in cycloaddition reactions with cyclic nitrones 2-5 was studied. The high regio- and stereoselectivity observed, sensibly increased by nonpolar solvents, was the effect of a double-asymmetric induction produced by the nitrone substituent on the pseudopeptidic tether. A new class of potent human tachykinin NK-2 receptor ligands was synthesized.
Dipeptides↗
Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals.
In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5' neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.
5' Untranslated Regions↗
Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box.
The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.
Adaptation, Physiological↗
Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions.
The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).
Bacterial Proteins↗
The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending.
Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.
Amino Acid Sequence↗
Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response.
A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.
Adaptation, Physiological↗
Post-transcriptional regulation of CspA expression in Escherichia coli.
The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter. After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.
Bacterial Proteins↗
Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression.
Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from -282 to +25 with two sites, closely flanking the DNA bend located at -150 from the transcriptional startpoint, partly overlapping the H-NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H-NS and the hns promoter region were studied by examining the effects of FIS and H-NS on in vitro transcription of hns-cat fusions, as well as looking at the effect of FIS on preformed complexes containing H-NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H-NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns.
Bacterial Outer Membrane Proteins↗
Interaction of the main cold shock protein CS7.4 (CspA) of Escherichia coli with the promoter region of hns.
Escherichia coli protein CS7.4 (CspA), homologous to the class of eukaryotic Y-box DNA-binding proteins, is a cold shock transcriptional activator of at least two genes, hns and gyrA. It was demonstrated that all or nearly all the elements necessary for the stimulation of hns transcription by CS7.4 protein are located in the proximal 110 bp DNA fragment of this gene with no additional elements being present in a longer fragment (660 bp) extending further upstream from the hns promoter. Protein CS7.4 bound strongly to the 110 bp segment of the hns promoter in crude extracts of cold shocked cells, but the purified protein displayed a weak interaction with the same DNA fragment. Purified CS7.4 protein also caused increased or decreased accessibility to DNase I at different sites of the 110 bp fragment of hns but the majority of these effects was seen only in the presence of RNA polymerase. Since gel shift experiments showed that protein CS7.4 stimulated the binding of RNA polymerase to the promoter of hns and since it is known that there are similarities between CS7.4 and ssDNA-binding proteins, we suggest that formation of the open complex by the RNA polymerase or protein-protein contacts between CS7.4 and the RNA polymerase are prerequisites for and/or the effects of the interaction of CS7.4 with its DNA target. The presence of a conserved CCAAT element in the hns promoter region, on the other hand, was found not to be stringently required for cold shock activation since expression of E coli of an hns-cat fusion containing the Proteus vulgaris hns promoter lacking a CCAAT box increased over four-fold after cold shock.
Bacterial Proteins↗
Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS.
The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion.
Bacterial Proteins↗
[Yersinia enterocolitica: biotypes and serotypes isolated from horticultural products].
A survey has been carried out for the presence of Yersinia enterocolitica (Y.e.) in horticultural products. 100 samples of these vegetables have been examined among the families of Compositae (lettuce, prickly lettuce, cabbage lettuce, common chicory, artichoke), Umbrelliferae (curly parsley, carrot, celery, fennel), Cruciferae (garden cabbage, red radish), Liliaceae (onion), and Solanaceae (tomato). 12 strains have been recorded in carrots (serotype 0:6.30), 1 strain in curly parsley (0:4.32), 3 in celery (0:7.8 and 0:6.30), and 1 in red radish (NAG). 0:6.30--the most commonly serotype found in the horticultural products--can be of special significance in human pathology, since the reference strain (IP 102) has been first isolated from man; besides, 0:6.30 has been recovered from four cases of human infection, and represents the most frequently isolated serotype from humans in Canada, together with 0:3, 0:5.27 and 0:9. According to other Authors, in can be assumed that "environmental" strains of Y.e. may also acquire virulence and cause infection in man. Moreover, a high number of environmental biotypes in foods may mask the presence and recovery of such strains involved in human pathology.
Food Microbiology↗
[Effect of L dopa, dopamine and CB 154 on prolactin secretion from human pituitary adenomas in vitro (proceedings) (author's transl)].
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Adenoma↗
Organ culture of human somatotrophic pituitary adenomas: ultrastructure and growth hormone production.
Ten somatotrophic adenomas removed from acromegalic patients and fragments of the non-tumoural surrounding pituitary were submitted to organ culture for periods of up to one month. Electron microscopic observation shows that these tumours retain their histological differentiation throughout the culture period. The cell morphology of the cultured tumours remains essentially unchanged and in particular the secretory granules keep their initial size (150 and 130 nm). However, the granules disappear gradually so that most of the cells look chromophobic by the 4th week of culture, and numerous lysosomes as well as autophagic figures appear at the same time. The hGH concentration in the culture medium has been measured of 4 adenomas. It is very high (10-fold greater than that from non-tumoural pituitary medium) during the first week (range 200-300 mug/ml). It still remains very high in the same experiments until the second week of culture in one experiment (200 mug/ml). After incubation of cultures with 3-H-leucine 3 H-hGH is obtained in the medium giving evidence of hormone synthesis by adenoma cells in culture. 3-H-hGH represents 40 percent of 3-H-proteins in the culture medium and gives the same elution pattern as standard hGH on Sephadex G 100 chromatography. A certain degree of correlation is observed between morphological and biological results: the greatest hGH production is obtained from explants which maintain the best histological appearance.
Acromegaly↗
[Effect of SRIF (somatostatin) on human somatroptropic adenomas in vitro ultrastructure and growth hormone production (author's transl)(proceedings)].
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Adenoma↗
[The content and structure of nursing documentation in Careggi Hospital, Florence, 1998: results and perspectives].
This retrospective, observational study was performed to evaluate the structure and the content of the nursing documentation in the Azienda ospedaliera Careggi, Firenze in 1998. To this aim we review 1964 nursing records including both notes by turns and care plans. One-thousand-one-hundred-and-twenty-five records came from surgical and 839 from medical wards. From the selected records, every day of the hospital stay, including both the admission and the discharge, were evaluated, so that the studied days were a total of 18,683. Only 32% of the nursing records had a global assessment of patient situation on admission. A medical diagnosis was observed in 84% of the cases, but a nursing diagnosis was absent in over 99.5% of the charts. During stay most notes were related to medical treatment and visits. Nursing notes were lacking in 32% of turns, while "nothing to report" was recorded in another 15.5% of cases. A nursing care plan was present in a minority of records. A final evaluation of planned nursing interventions was reported in approximately 5% of the charts. Nursing care plans were updated during stay in less than one tenth of cases. Discharges notes were absent in slightly more than 80% of the cases. This survey confirms the importance of continuing education and supervision in nursing documentations, if a reliable source of nursing information has to be developed. Future nursing records should include only essential information, avoiding any overlap with medical charts.
Adolescent↗