Investigating the effects of the local environment on bottlebrush conformations using super-resolution microscopy.

The single-chain physics of bottlebrush polymers plays a key role in their macroscopic properties. Although efforts have been made to understand the behavior of single isolated bottlebrushes, studies on their behavior in crowded, application-relevant environments have been insufficient due to limitations in characterization techniques. Here, we use single-molecule localization microscopy (SMLM) to study the conformations of individual bottlebrush polymers by direct imaging. Our previous work focused on bottlebrushes in a matrix of linear polymers, where our observations suggested that their behavior was largely influenced by an entropic incompatibility between the bottlebrush side chains and the linear matrix. Instead, here we focus on systems where this effect is reduced: in solvent-swollen polymer materials and in systems entirely composed of bottlebrushes. We measure chain conformations and rigidity using persistence length (lp) as side chain molecular weight (Msc) is varied. Compared to a system of linear polymers, we observe greater flexibility of the backbone in both systems. For bottlebrushes in bottlebrush matrices, we additionally observed a scaling relationship between lp and Msc that more closely follows theoretical predictions. For the more flexible chains in both systems, we reach the edge of our resolution limit and cannot visualize the entire contour of every chain. We bypass this limitation by discussing the aspect ratios of the features within the super-resolution images.

Direct visualization of bottlebrush polymer conformations in the solid state.

Although the behavior of single chains is integral to the foundation of polymer science, a clear and convincing image of single chains in the solid state has still not been captured. For bottlebrush polymers, understanding their conformation in bulk materials is especially important because their extended backbones may explain their self-assembly and mechanical properties that have been attractive for many applications. Here, single-bottlebrush chains are visualized using single-molecule localization microscopy to study their conformations in a polymer melt composed of linear polymers. By observing bottlebrush polymers with different side chain lengths and grafting densities, we observe the relationship between molecular architecture and conformation. We show that bottlebrushes are significantly more rigid in the solid state than previously measured in solution, and the scaling relationships between persistence length and side chain length deviate from those predicted by theory and simulation. We discuss these discrepancies using mechanisms inspired by polymer-grafted nanoparticles, a conceptually similar system. Our work provides a platform for visualizing single-polymer chains in an environment made up entirely of other polymers, which could answer a number of open questions in polymer science.

Evidence-Based Practices to Promote Tobacco Cessation During Pregnancy in a Sample of Romanian General Practitioners.

Changes in confidence in implementing smoking cessation support for pregnant women was assessed among Romanian General Practitioners (GPs) before and after a training program of evidence-based clinical practices to promote quitting. The total number of physicians participating in the study was 69. Before training, 51% of GPs felt somewhat/very confident asking pregnant women about tobacco use, 39% assisted smokers with a quit plan, 38% arranged follow-up for patients. After training, 85-90% found the training informative/very informative on: how to ask patients if they smoke (89%), advising patients to quit (88%), talking about the benefits of quitting (85%), assessing patients readiness to quit (87%), assisting patients in setting a quit date (87%).

Dynamic Jahn-Teller effect for V²+ in MgO single crystal.

In this paper we studied the dynamic Jahn-Teller effect in the excited state (4)T(2g) of the V(2+) ions doped in MgO crystal, in octahedral coordination. Calculation of the fine structure of the energy levels in the frame of crystal field theory, estimation of the vibronic coupling constants and the Jahn-Teller stabilization energy are presented. The dynamic Jahn-Teller effect in the lowest excited (4)T(2)(g) state of the V(2+) ions is considered using the Ham effect and analysis of the displacements of ligands, due to the combined effect of the a(1)(g) and e(g) normal modes of the obtained octahedral cluster.

Crystal field analysis of energy level structure of LiAlO2:V3+ and LiGaO2:V3+.

A detailed analysis of the energy level structure of tetrahedrally coordinated V(3+) ion in lithium aluminum oxide LiAlO(2) (gamma-phase) and lithium dioxogallate LiGaO(2) is performed using the exchange charge model of the crystal field theory. The parameters of the crystal field acting on the V(3+) optical electrons are calculated from crystal structure data assuming C(1) point symmetry of the [VO(4)](5-) impurity center in LiAlO(2) and LiGaO(2). Crystal field splitting of all five LS terms of the V(3+) ion ((3)F, (3)P, (1)S, (1)D, (1)G) is calculated. The energy levels obtained are compared with experimental absorption spectra and results of application of other crystal field models (the angular overlap model and Racah theory) to the considered crystals; though only one fitting parameter of the exchange charge model was used, a good agreement with experimental data on the ground and excited state absorption is demonstrated.

Regulation of the initial segment of the murine epididymis by dihydrotestosterone and testicular exocrine secretions studied by expression of specific proteins and gene expression.

The murine caput epididymidis responded to deprivation of luminal fluid from the testis by regression of the initial segment but maintenance of the adjacent proximal and distal caput regions, as judged by immunohistochemical staining of the glutamate transporter EAAC1 and the lipocalin MEP17 and enzymatic activity of beta-galactosidase (beta-Gal). Additional removal of circulating androgens by bilateral castration similarly led to loss of the initial segment and of the proximal caput but the distal caput was transformed into an epithelium containing more apical than principal cells staining for EAAC1; this epithelium resembled the precursor epithelium usually only seen in prepubertal juveniles. Administration of dihydrotestosterone (DHT) to the castrates maintained the proximal and distal caput epithelia and induced a proximal epithelium, which resembled the initial segment in its prominent staining for Golgi, EAAC1 and beta-Gal activity, although it was short and exhibited no MEP17 expression. DHT was present in the c-ros knockout caput epididymidis lacking the initial segment and in the heterozygous organ but the DHT concentration was lower in the knockout corpus. The maintenance of the full complement of epithelia in the murine caput epididymidis in the adult thus requires a combination of luminal fluid from the testis, tissue DHT and the presence of the c-ros oncogene.

Development of the caput epididymidis studied by expressed proteins (a glutamate transporter, a lipocalin and beta-galactosidase) in the c-ros knockout and wild-type mice with prepubertally ligated efferent ducts.

Expression of a glutamate transporter (EAAC1), a lipocalin (MEP17) and beta-galactosidase (beta-Gal) in histological sections was used to monitor post-natal development of the murine epididymis. Three epithelia in the adult caput of wild-type mice were distinguished: I, the initial segment; II, the proximal caput; and III, the distal caput. The regions in which epithelia I, II and III were situated were called regions I, II and III, respectively. Regions I, II and III developed from a precursor epithelium present on day 14; from day 16, a presumptive region I epithelium was evident and, by day 21, epithelia characteristic of future regions II and III appeared. The relationship between the c-ros gene and the initial segment was studied by investigating the development of the caput epididymidis in transgenic homozygous c-ros knockout (-/-) mice that lack the initial segment, heterozygous (+/-) males and wild-type males in which the efferent ducts had been ligated prepubertally so that the initial segment failed to develop. In mice with prepubertally ligated efferent ducts, regions II and III developed normally but region I was missing in the adult and expression of c-ros was partially decreased. In (-/-) mice, the precursor epithelium was present, differentiation of epithelium II was delayed until day 32 and epithelium I never developed. Thus, caput region I develops before c-ros expression, high testosterone secretion and differentiation of regions II and III but not if the organ is deprived of the oncogene c-ros or testicular exocrine secretions. The caput of the knockout male lacks solely the initial segment so that the efferent ducts are in continuity with the post-initial segment, proximal caput region. The ligand for c-ros may be present in testicular fluid and both ligand and receptor may be necessary for differentiation of epithelia I and II.

Emergency Health Care Information System for Bucharest-Romania.

The Emergency Health Care Information System (EHCIS) in Bucharest provides information about the whole activity of Dispatch Emergency Ambulance Service and Emergency Receiving Room of the 7 Hospitals, providing emergency health care in Bucharest over a MAN (Metropolitan Area Network). In each of these places a local network is located, containing a database server ORACLE. The link among LANs is made via switched lines. The Hospitals collect information only about emergency cases. The microstation represents station for emergency teams of Emergency Ambulance Service of Bucharest (EASB), distributed in all 6 districts of Bucharest. The system is structured accordingly with the working-groups existing in Dispatch, microstations and hospitals: registration operators (phone-operators) for administer the emergency requests/calls; a location for the medical coordinator which must to choose, in few seconds, the emergency team, accordingly with the case emergency degree; radio-operators which communicate with the teams in the field; a location for the manager of Dispatch, in order to provide a full-set of real-time medical and resources information; a registration operator at each microstation; a registration operator at each hospital. The data are registered in the ORACLE database on the central server. The client/server architecture assures the real time communication among all these locations. The system works 7 days/week, 24 hours/day.

[Evaluation in adolescence of patients treated during their childhood and the prevention of psychoses]. / L'évaluation à l'adolescence de patients traités pendant leur enfance et la prévention des psychoses.

The Evening Care unit is designed for 30 children and adolescents who present severe disturbances in their psychological development and who live in perturbed-families. Results are not assessed against symptom evolution or behavior modification criteria but by studying changes in mental functioning and subject's capacity to work through the adolescent ruptures. This assessing process is illustrated with three particularly difficult case presentations.

Hepatic lipase: a member of a family of structurally related lipases.

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.

Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.

CCK-5: sequence analysis of a small cholecystokinin from canine brain and intestine.

The purpose of this study is to purify and to characterize chemically cholecystokinin (CCK)-like peptides present in brain and gut extracts that elute from gel filtration after the octapeptide. Canine small intestinal mucosa and brain were boiled in water and then extracted in cold trifluoroacetic acid, and cholecystokinin-like immunoreactivity was determined by carboxyl-terminal specific radioimmunoassay. Gel permeation chromatography on Sephadex G-50 revealed a form of CCK apparently smaller than CCK-8. This peptide was purified by immunoaffinity chromatography and three successive reverse phase high-performance liquid chromatography steps. Microsequence analysis showed that the amino terminal primary sequence of this small CCK was Gly-Trp-Met-Asp. Immunochemical and chromatographic analysis indicated that the carboxyl-terminal residue was Phe-NH2 and thus the full sequence is Gly-Trp-Met-Asp-Phe-NH2. An antibody that recognizes synthetic CCK-8, CCK-5, and CCK-4 equally did not reveal the presence of significant amounts of CCK-4. These results indicate that CCK-5 is the major CCK form smaller than the octapeptide present in brain (19% of total CCK immunoreactivity) and small intestine (7% of total). This finding, coupled with the demonstration by others that CCK-5 interacts with high-affinity brain CCK receptors, indicates that CCK-5 may play a physiological role in brain function.

New molecular forms of cholecystokinin. Microsequence analysis of forms previously characterized by chromatographic methods.

Cholecystokinin previously has been characterized by chromatographic and immunological techniques. Analysis of cDNA has revealed the structure of preprocholecystokinin. The predicted processing sites of preprocholecystokinin cannot account for the multiple forms of cholecystokinin detected. This report details the isolation and characterization of cholecystokinin peptides containing 58, 39, 33, 25, 18, 8, 7, and 5 amino acids. None of the cleavages that occurred to form these peptides was at the carboxyl side of double basic residues. Cholecystokinin 25, 18, and 7 have not previously been isolated and identified by sequence analysis. The processing that forms these peptides includes cleavage after single basic residues for the 58, 39, 33, and 8 amino acid peptides. The 25, 18, 7, and 5 amino acid peptides must be formed by other endopeptidases or combinations of endo- and exopeptidases. The analysis of this series of peptides provides the chemical basis for structural differences in cholecystokinin molecules previously demonstrated by chromatographic and immunological methods. These structures provide insight into tissue-specific processing that occurs for this important regulatory peptide in intestine and brain.

Isolation and characterization of a neurotensin-like decapeptide from a canine upper small intestinal extract.

Neurotensin-like immunoreactivity can be detected in extracts of canine upper gastrointestinal mucosa when measured by carboxyl terminal but not by amino terminal antibodies to neurotensin. The nature of this immunoreactive material was characterized by complete purification on gel filtration and HPLC followed by peptide microsequence analysis. The structure obtained was Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-(Leu), identical in structure to the carboxyl terminal decapeptide of neurotensin. It cannot, however, be excluded that this neurotensin decapeptide was generated from a larger neurotensin-like peptide during the extraction procedure by a physiological or artificial enzymatic process. Since carboxyl terminal neurotensin fragments containing eight or more residues have full biological activity, this peptide may be responsible for neurotensin-like biological activities within the mucosa of, or after release from, the upper gut.

Homology of lipoprotein lipase to pancreatic lipase.

Bovine milk lipoprotein lipase was subjected to amino acid sequence analysis. The first 19 amino-terminal residues were Asp-Arg-Ile-Thr-Gly-Gly-Lys-Asp-Phe-Arg-Asp-Ile-Glu-Ser-Lys-Phe-Ala-Leu- Arg. In addition, reversed-phase high-performance liquid chromatography of a tryptic digest of reduced and alkylated lipase resolved a number of peptides, five of which contained cysteine. Sequence analysis of the tryptic peptides revealed in most instances a close homology to porcine pancreatic lipase. Based on this homology, the relative alignment of the sequenced lipoprotein lipase peptides can be made. In addition, a potential binding site for the triacylglycerol substrate and a carbohydrate-binding domain for lipoprotein lipase are postulated.

Amino-terminal amino acid sequence of murine colony-stimulating factor 1.

We report the amino acid composition, the NH2-terminal sequence, and the tryptic map by HPLC for murine L-cell colony-stimulating factor 1 (CSF-1). CSF-1 purified by affinity chromatography was S-carboxymethylated under denaturing conditions and subjected to sequence microanalysis. CSF-1 contains four or five cysteine residues per subunit and approximately 30-40% carbohydrate by weight. Three contiguous cysteine residues were located in the NH2-terminal sequence. An additional two cysteine residues were located on the tryptic map. Previous studies as well as immunoblotting studies reported here suggest that sulfhydryl bridging plays a major role in maintaining the conformation of the protein. Carbohydrate was located on two tryptic fragments. The findings of a single NH2-terminal sequence in high yield and a relatively simple tryptic map (15 major peptides) suggest that CSF-1 contains two identical subunits. The NH2-terminal sequence of CSF-1 has only limited homology to insulin or insulin-like hormones but no homology with granulocyte/macrophage-CSF or interleukin 3.

Rapid high-yield purification of canine intestinal motilin and its complete sequence determination.

Canine motilin has been purified from small amounts of canine intestine in a form suitable for microsequence analysis. The sequence determined is: Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg- Asn-Lys - Ile-Arg-Asn-Lys-Gly-Gln. Canine motilin differs from porcine motilin at five positions. The rapid, high-yield (24% overall yield) microisolation techniques used for canine motilin should be suitable for the isolation of other basic peptides found in low levels in tissue that is available only in limited amounts. These methods should make the isolation and sequence determination of human brain and gut peptides more readily achievable.

Primary structure of a glycosylated DNA-binding domain in human plasma fibronectin.

The complete amino acid sequence of a DNA-binding domain isolated from human plasma fibronectin after limited trypsin digestion has been obtained. It contains 132 amino acids and one biantennary glycosyl unit at residue 104, for an estimated Mr of 16,931. The fragment can be purified by a two-step procedure consisting of DNA-affinity chromatography and reverse-phase high performance liquid chromatography. It can also be purified by heparin-affinity chromatography. The domain is unusual in its susceptibility to tryptic-like cleavages even by neutral or aromatic residue-specific proteases. It has no cysteine residues and is predicted to favor a beta-sheet structure by Chou and Fasman analysis. Based on this analysis we have proposed a model which exhibits a clustering of aromatic and basic residues, consistent with similar involvement of basic and aromatic residues in other DNA-binding proteins. The net charge of the domain at neutral pH (+1, without sialic acid) argues against a nonspecific charge interaction with polyanionic macromolecules such as DNA and heparin. Internal sequence repeats occur at intervals of 30, 60, and 90 residues, thus suggesting a maximum size for a repetitive building block which gave rise to this domain.

Isolation and sequence analysis of human bombesin-like peptides.

The decapeptide form of human gastrin releasing peptide was isolated from acid extracts of liver tissue containing a metastatic human bronchial carcinoid tumor. A larger form also was isolated and partially characterized. During gel permeation chromatography the major immunoreactive peak eluted in the same region as synthetic gastrin releasing decapeptide while a second minor immunoreactive peak eluted near gastrin releasing peptide. Bombesin-like immunoreactivity (BLI) was purified by successive applications to reverse phase high pressure liquid chromatography (HPLC) columns. After four successive HPLC purifications a single peak of bombesin-like immunoreactivity was detected. Amino acid analysis, microsequence analysis and coelution with synthetic peptide indicated that the predominant form present in metastatic tumor tissue was identical to the decapeptide form of canine gastrin-releasing peptide. The less abundant form was purified by cation exchange chromatography followed by reverse phase high pressure liquid chromatography. Partial microsequence analysis of this peptide, through the first 11 residues, was Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu. This sequence differed from that of hog heptacosapeptide gastrin releasing peptide at positions 1,3,4 and 5 and from the canine peptide as positions 1,3,5, and 7.