Conformational Studies of PACAP(1–27) and Its Fragments

Summary Conformational features of the neuropeptide pituitary adenylate cyclase activating polypeptide (1–27) (PACAP(1–27)) and its shorter fragments (1–5), (7–11) and (14–27) were studied by circular dichroism (CD) and fluorescence spectroscopy. The obtained CD spectra revealed that only PACAP(1–27) and the fragment (14–27) possess some content of an organized structure — the α-helix. This C-terminal, helical part of the peptides is important for receptor binding as it provides a stable structure that can reside in the ordered lipid region of the receptor site in the membrane, while the primary biological function of the hormone resides in the N-terminal, disordered part. Fluorescence studies have revealed that the tyrosine residue located in the helical region of PACAP has a higher quantum yield and a longer average lifetime than the tyrosine in the N-terminus, probably due to a ‘shielding’ effect of the hydrophobic cluster around Tyr²².     

Neo-Epitopes—Fragments of Cartilage and Connective Tissue Degradation in Early Rheumatoid Arthritis and Unclassified Arthritis

Objective

Tissue destruction in rheumatoid arthritis (RA) is predominantly mediated by matrix metalloproteinases (MMPs), thereby generating protein fragments. Previous studies have revealed that these fragments include MMP-mediated collagen type I, II, and III degradation, citrullinated and MMP-degraded vimentin and MMP degraded C-reactive protein. We evaluated if biomarkers measuring serum levels of specific sequences of the mentioned fragments would provide further information of diagnostic and/or prognostic processes in early arthritis.

Methods

Ninety-two early arthritis patients (arthritis duration<1 year, DMARD naïve) were enrolled. Patients either fulfilled the ACR/EULAR2010 criteria for RA (n = 60) or had unclassified arthritis (UA) (n = 32). Patients fulfilling the RA criteria after 2 years follow-up were classified into non-erosive (n = 25), or erosive disease (n = 13). Concentrations of the biomarkers: C1M, C2M, C3M, VICM and CRPM were measured in baseline serum.

Results

C1M, C3M and CRPM were able to discriminate between the UA and RA baseline diagnosis in 92 patients with an AUROC of 0.64 (95%CI 0.517 to 0.762), 0.73 (95%CI 0.622 to 0.838) and 0.68 (95%CI 0.570 to 0.795). C2M showed a potential for discrimination between non-erosive and erosive disease in 38 patients with an AUROC of 0.75 (95%CI 0.597 to 0.910). All of the applied biomarkers correlated with one or more of the disease activity parameters: DAS28, ESR, CRP, SJC66, TJC68 and/or HAQ.

Conclusion

This is the first study evaluating the applied biomarkers at this early stage of arthritis. C1M, C3M, CRPM might be the best diagnostic marker, whereas high levels of C2M indicated progression of disease at follow-up in early RA patients.     

Fragments d’anthropologie clinique en action

The human condition means that both humaneness and inhumanity dwell in human beings, along with light and shadow, madness and reason. This ‘holistic’ conception of human life, which is based on the notion of drive, is under relentless threat from objectifying, psychiatric reductionism. Such impoverishment of the psyche is evident in neuroscience, sociologism and economism; the psyche is the locus of fragility, which constitutes the human condition and flows from the precarious balance between shadow and light in every person’s life. Psychiatry draws on the tendency of today’s society to eradicate evil, or at least make it invisible. This results in a world of hypertransparency, where negativity is obscured. Hence, life is deprived of destiny, of its tragic dimension, and of transcendence; it is under general anaesthesia, with no apparent suffering or pain. An anthropoclinical approach, which views human beings in their totality, offers an alternative to psychiatry’s objectification of the psyche. It goes beyond the alliance of the therapeutic and scientific and is driven by the ethical concern of a genuinely anthropological and clinical approach that allows meeting the Other in an existential union.     

Structural Flexibility of CaV1.2 and CaV2.2 I-II Proximal Linker Fragments in?Solution

Voltage-dependent calcium channels (Ca_V) enable the inward flow of calcium currents for a wide range of cells. Ca_V1 and Ca_V2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the Ca_Vβ subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the Ca_V subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the Ca_Vβ-binding site, called the proximal linker (PL). The results demonstrate that the Ca_V1.2 PL is more flexible than the Ca_V2.2 PL, the flexibility is intrinsic and not dependent on Ca_Vβ binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role.     

Effects of Bovine β-Casein (1-28) and Its Chemically Synthesized Partial Fragments on Proliferative Responses and Immunoglobulin Production in Mouse Spleen Cell Cultures

Effects of bovine β-casein (1-28) having a phosphoserine-rich region (Glu¹⁴-SerP-Leu-SerP-SerP-SerP-Glu-Glu²¹) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1-28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG+IgM+IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated β-casein (14-21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine β-casein (1-28), but β-casein (14-21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides in attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.     

The Structure of Aβ42 C-Terminal Fragments Probed by a Combined Experimental and Theoretical Study

The C-terminus of amyloid β-protein (Aβ) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Aβ42 have been shown to disrupt oligomerization and to strongly inhibit Aβ42-induced neurotoxicity. Here we study the structures of selected CTFs [Aβ(x-42); x = 29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable β-structure: β-hairpin for Aβ(x-42) (x = 29-31) and extended β-strand for Aβ(39-42). The β-hairpin of Aβ(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the β-hairpin in Aβ42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of β-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.     

Heterocyclic Resveratrol Analogs. Synthesis and Physiological Activity: Part 1–Analogs Obtained by the Replacement of Aryl Residues with Heterocyclic Fragments

Russian Journal of Bioorganic Chemistry - Resveratrol is a polyphenolic phytoalexin, a stilbene derivative, whose physiological activities have been studied in a large number of studies. It...     

The Immune Response to Artificial Proteins Containing Biologically Active Fragments of Human IFN-α2 and Insulin

A study was made of the humoral immune response of BALB/c mice to various doses of artificial proteins that contained biologically active fragments of human interferon 2 (IFN-2) and insulin. The insulin fragment had no effect on the response to any protein construct. The IFN-2 fragment increased the titer of antibodies against the construct. Mapping of continuous B epitopes with immune sera revealed several antigenic determinants, the C end of the IFN-2 fragment with the adjacent de novo protein region being immunodominant. The more effective binding of serum antibodies with the constructs containing the IFN-2 fragment was attributed to antibody interaction with the fragment and to better recognition of the entire protein construct by the immune system.     

Synthesis of RNA Fragments Using the H-Phosphonate Method and 2′-(2′-Chlorobenzoyl) Protection

Abstract

The performance of 2′-(2-chlorobenzoyl) protected ribonucleoside H-phosphonates in the synthesis of oligoribonucleotides has been studied.     

Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

By means of agarose-gel electrophoresis, endonuclease R.EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, phi80, and hybrid phages were constructed.     

Botulinum Neurotoxin Type A: Limited Proteolysis by Endoproteinase Glu-C and α-Chymotrypsin Enhanced Following Reduction; Identification of the Cleaved Sites and Fragments

Botulinum neurotoxin (NT) serotype A is a ～150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1–Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1–Lys 437 and Ala 448–Leu 1295 in the ～50-kDa light (L) and ～100-kDa heavy (H) chains, respectively, connected by a Cys 429–Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49–57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423–472]. Endoproteinase Glu-C and α-chymotrypsin were used for controlled digestion at pH 7.4 of the ～150-kDa dichain NT and the isolated ～100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in ～30- and ～20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249–Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193–1200] to undergo “self-cleavage” following reductive separation of the L chain from the H chain. The site Tyr–Tyr–Glu–Met (residues 249–252) appears to become exposed following reduction of Cys 429–Cys 453 disulfide. Identification of Glu 669–Ile 670 and Tyr 683–Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659–684), part of which (residues 659–681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci. 4, 1490–1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure–function studies; the ～85- and ～45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.     

Nonreplicated DNA and DNA Fragments in T4 r? Bacteriophage Particles: Phenotypic Mixing of a Phage Protein

"Conservative phage" containing a genome derived from an infecting phage particle which has not undergone replication in the cell but nevertheless has become encapsulated and released in a normal phage particle, are found after infection of Escherichia coli with rII(-) or rI(-) mutants under conditions which result in rapid lysis. If such conservative phage are derived from a mixed infection with v(+) and v(1) phage, they display phenotypic mixing of the v gene product (an endonuclease carried in the phage particle). Populations of rI and rII mutant phage grown under conditions of rapid lysis include particles containing short DNA fragments. It is suggested that a "maturation defect", common to rI and rII mutants, but absent in rIII mutants, may account for the encapsulation of nonreplicated DNA as well as that of the DNA fragments.     

Mapping of New Escherichia coli K and 15 Restriction Sites on Specific Fragments of Bacteriophage φX174 DNA

We have isolated several new phiX174 mutants which contain sites sensitive to restriction by Escherichia coli. One contains an E. coli 15 restriction site and three are double mutants containing an E. coli K site as well as the E. coli 15 site. The replicative form (RF) DNA of one of the mutants containing a K site has been shown to be restricted in spheroplasts of a K-12 strain. The infectivity of this RF, but not wild-type RF, has also been shown to be inactivated by an E. coli K extract and by purified K restriction enzyme in vitro. The product of the RF treated with purified K restriction enzyme in vitro is a full length linear molecule. The mutant sites have also been localized to specific regions of the phiX174 genome by a fragment mapping technique, making use of specific fragments of phiX174 RF DNA obtained by digestion with a specific endonuclease.     

Biological Relevance of Natural α-Toxin Fragments from Staphylococcus aureus

Serine proteases represent an essential part of cellular homeostasis by generating biologically active peptides. In bacteria, proteolysis serves two different roles: a major housekeeping function and the destruction of foreign or target cell proteins, thereby promoting bacterial invasion. In the process, other virulence factors such as exotoxins become affected. In Staphylococcus aureus culture supernatant, the pore-forming α-toxin is cleaved by the coexpressed V8 protease and aureolysin. The oligomerizing and pore-forming abilities of five such spontaneously occurring N- and C-terminal α-toxin fragments were studied. ³H-marked α-toxin fragments bound to rabbit erythrocyte membranes but only fragments with intact C termini, missing 8, 12 and 71 amino acids from their N-terminal, formed stable oligomers. All isolated fragments induced intoxication of mouse adrenocortical Y1 cells in vitro, though the nature of membrane damage for a fragment, degraded at its C terminus, remained obscure. Only one fragment, missing the first eight N-terminal amino acids, induced irreversible intoxication of Y1 cells in the same manner as the intact toxin. Four of the isolated fragments caused swelling, indicating altered channel formation. Fragments missing 12 and 71 amino acids from the N terminus occupied the same binding sites on Y1 cell membranes, though they inhibited membrane damage caused by intact toxin. In conclusion, N-terminal deletions up to 71 amino acids are tolerated, though the kinetics of channel formation and the channel’s properties are altered. In contrast, digestion at the C terminus results in nonfunctional species.     

The Occurrence of Matriptase C-Terminal Fragments on the Apical and Basolateral Sides of Madin–Darby Canine Kidney Epithelial Cells

Matriptase is a type II transmembrane serine protease. In the present study, matriptase C-terminal fragments containing the catalytic serine protease domain were found to occur on the apical and basolateral sides of Madin–Darby canine kidney epithelial cells transfected with a cDNA encoding the protease. This suggests that matriptase interacts with various potential substrates when expressed in simple epithelia.     

A Survey of λ Repressor Fragments from Two-State to Downhill Folding

We survey the two-state to downhill folding transition by examining 20 λ_6-85^? mutants that cover a wide range of stabilities and folding rates. We investigated four new λ_6-85^? mutants designed to fold especially rapidly. Two were engineered using the core remodeling of Lim and Sauer, and two were engineered using Ferreiro et al.'s frustratometer. These proteins have probe-dependent melting temperatures as high as 80 °C and exhibit a fast molecular phase with the characteristic temperature dependence of the amplitude expected for downhill folding. The survey reveals a correlation between melting temperature and downhill folding previously observed for the β-sheet protein WW domain. A simple model explains this correlation and predicts the melting temperature at which downhill folding becomes possible. An X-ray crystal structure with a 1.64-Å resolution of a fast-folding mutant fragment shows regions of enhanced rigidity compared to the full wild-type protein.     

Channel-Forming Abilities of Spontaneously Occurring α-Toxin Fragments from Staphylococcus aureus

Pore formation by four spontaneously occurring α-toxin fragments from Staphylococcus aureus were investigated on liposome and erythrocyte membranes. All the isolated fragments bound to the different types of membranes and formed transmembrane channels in egg-phosphatidyl glycerol vesicles. Fragments of amino acids (aa) 9–293 (32 kD) and aa 13–293 (31 kD) formed heptamers, similar to the intact toxin, while the aa 72–293 (26 kD) fragment formed heptamers, octamers, and nonamers, as judged by gel electrophoresis of the liposomes. All isolated fragments induced release of chloride ions from large unilamellar vesicles. Channel formation was promoted by acidic pH and negatively charged lipid head groups. Also, the fragments’ hemolytic activity was strongly decreased under neutral conditions but could be partially restored by acidification of the medium. We paid special attention to the 26-kD fragment, which, despite the loss of about one-fourth of the N-terminal part of α-toxin, did form transmembrane channels in liposomes. In light of the available data on channel formation by α-toxin, our results suggest that proteolytic degradation might be better tolerated than previously reported. Channel opening could be inhibited and open channels could be closed by zinc in the medium. Channel closure could be reversed by addition of EDTA. In contrast, digestion at the C terminus led to premature oligomerization and resulted in species with strongly diminished activity and dependent on protonation.