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At least 289 records · Page 16Linked to original sources
PubMed1994

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA.

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hylA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E. coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

Antibodies, Bacterial↗
PubMed1994

Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy: a comparative study with C-terminal(109-141)- and N-terminal(1-86)-region-specific PTHrP radioassay.

We evaluated the circulating forms of immunoreactive PTHrP in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1: 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) HHM patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1: 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r = 0.86). The values also showed a close correlation with serum Ca; r = 0.75 for C-RAS and r = 0.81 for N-RAS. In addition, the correlations between the PTHrP reading obtained with the different RASs and serum Cr were: r = 0.42 with C-RAS and r = 0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP(109-141). In contrast, circulating concentrations of the PTHrP(1-86) fragment are very low, but detection of the PTHrP(1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PTHrP(109-141) fragment with C-RAS.

Adolescent↗
PubMed1988

Ultrastructural differences between pallidohabenular terminals and pallidothalamic terminals.

Pallidal terminals in the cat lateral habenular nucleus and the ventral anterior and ventral lateral nuclear complex of the thalamus have been examined electron microscopically. A significant difference was obtained after a quantitative analysis of size and shape parameters of synaptic vesicles in the identified terminals: synaptic vesicles in the pallidohabenular terminals had smaller mean elongation index and larger mean vesicle profile area than those in the pallidothalamic terminals.

Animals↗
PubMed1989

Dynorphin A(1-8) immunoreactive cell bodies, dendrites and terminals are postsynaptic to calcitonin gene-related peptide primary afferent terminals in the monkey dorsal horn.

In the present study, Dynorphin A(1-8) (DYN)-immunoreactive cell bodies, dendrites and terminals are observed postsynaptic to calcitonin gene-related peptide (CGRP)-immunoreactive terminals in laminae I and II. With the demonstration of axosomatic, axodendritic as well as axoaxonic interactions, we hypothesize that CGRP-containing primary afferent terminals can effect processing at the level of the cell body (possibly influencing the manufacture of DYN and/or the firing rate of DYN-containing cells) and also modulate the output of DYN terminals in laminae I and II of the monkey dorsal horn. These structural relationships may play an important role in the processing of noxious input at the spinal cord level.

Animals↗
PubMed1979

Dissociation between drug-induced increases in nerve terminal and non-nerve terminal pools of GABA in vivo.

To examine whether increased GABA levels produced by n-dipropylacetate (DPA) and amino-oxyacetic acid (AOAA) are associated with nerve terminals, we compared the effect of these drugs on the GABA content of substantia nigra (SN) in rats in which the GABAergic afferent projections to SN had been unilaterally destroyed. In the SN largely devoid of GABAergic nerve terminals, AOAA (30 mg/kg) produced a 2-fold increase in GABA, whereas DPA (300 mg/kg) was without effect. Since DPA and AOAA both increased GABA to a similar extent in the intact SN, it appears that the GABA increase produced by DPA is associated with GABAergic nerve terminals, while AOAA primarily elevates GABA in non-nerve terminal components (neural perikarya and glial cells) which are not destroyed by our lesions.

Aminooxyacetic Acid↗
PubMed1992

Matrix assembly of recombinant fibronectin polypeptide consisting of amino-terminal 70 kDa and carboxyl-terminal 37 kDa regions.

Three different forms of recombinant human fibronectin polypeptides consisting of the amino-terminal 70 kDa region, the carboxyl-terminal 37 kDa region, or both, were expressed in mouse L cells. Although either the amino-terminal or the carboxyl-terminal region alone was only poorly incorporated into the extracellular matrix, the fused form of the polypeptide was highly capable of assembling into the matrix. These results indicate that matrix assembly of fibronectin requires both regions and can proceed in the absence of the type III repeats including the one containing the cell adhesive Arg-Gly-Asp sequence.

Animals↗
PubMed1985

Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues.

Amino acid sequence data and results from limited proteolytic digestion have been used to define the three-domain structure of intermediate filament proteins. A centrally located highly alpha-helical domain of about 310 residues well-conserved in sequence principles and length is flanked by the highly variable sequences of the non-alpha-helical headpiece and tailpiece. A direct involvement in filament formation of one or both terminal domains was previously proposed for desmin since chymotryptic removal of head and tailpiece provided a derivative unable to form filaments. In order to evaluate directly the importance of these regions we have prepared desmin derivatives lacking either the amino-terminal 67 (T-desmin) or carboxy-terminal 27 residues (L-desmin). Whereas the latter derivative is fully polymerization-competent the fragment lacking only the basic and arginine-rich headpiece cannot form filaments on its own and remains in a protofilamentous stage. These structures of T-desmin are not incorporated into filaments when mixed with protofilaments of desmin. If, however, the two proteins are mixed in 7 M-urea subsequent dialysis provides morphologically normal filaments containing T-desmin. The results suggest that at least certain hybrid protofilaments containing less than four headpieces are accepted in the filament. The removal of the 27 carboxy-terminal residues in L-desmin, although not interfering with filament formation, leads to a change in surface since filaments show lateral aggregation at 170 mM but not at 50 mM salt. The results are discussed in relation to current models of intermediate filament structure.

Amino Acid Sequence↗
PubMed1991

The C-terminal half of the preS1 region is essential for the secretion of human hepatitis B virus large S protein devoid of the N-terminal retention sequence.

A large surface protein of human hepatitis B virus was expressed from a mutated S gene in which both the reported retention sequence (aa 2-19) and the C-terminal half of the preS1 region (aa 66-117) were deleted. This retention sequence-free protein was not secreted from the monkey kidney cell line COS-M6 in transient expression assays. When this large S protein was overexpressed in the presence of the major S protein, the secretion of the latter was severely inhibited. Moreover, the presence of S protein in large abundance did not facilitate the secretion of the mutated large S protein at all, indicating that it was subject to more profound retention than the wild-type large S protein. The findings that, like the wild-type large S protein, this altered protein retained some of the properties of its C-terminal S domain, and that the preS1 region was still on its surface suggested that there was no extensive alteration of the polypeptide folding. The interpretation is favored that the C-terminal half of the preS1 region plays a crucial role in the secretion of the large S protein when its N-terminal retention sequence is not present.

Animals↗
PubMed1981

A Golgi analysis of the accessory optic fibers terminating in the medial terminal nucleus of the mouse accessory optic system.

Fibers of the accessory optic tract (AOT) terminating in the medial terminal nucleus (MTN) were observed in the mouse by the rapid Golgi method. The AOT fibers, which entered the MTN from its ventromedial aspect, were divided into thick and fine fiber groups, the thick fibers emitting many terminal collaterals of various calibers, and the fine ones generating fine terminal branches. The possibility exists that the retinal neurons sending AOT fibers to the MTN might be heterogeneous in nature.

Animals↗
PubMed1982

A new approach to quantitate the density and antigen contents of high densities of transmitter-identified terminals. Immunocytochemical studies on different types of tyrosine hydroxylase immunoreactive nerve terminals in nucleus caudatus putamen of the rat.

By means of a semi-automatic image analyzer plugged into an Apple II computer and suitable computer programs it is possible to analyze transmitter-identified nerve terminals. Thus, a densitometric approach is applied on the original photograph followed by systematic sampling which is carried out by means of a grating of circles. This procedure allows the study quantitatively of density and intensities of different types of densely packed tyrosine hydroxylase (TH) immunoreactive nerve terminals of the nuc. caudatus putamen. It is shown that the islandic TH immunoreactive nerve terminals have a higher density, but a TH content similar to the diffuse types of TH immunoreactive nerve terminals in the nuc. caudatus putamen.

Animals↗
PubMed2003

N-terminal insertion of alamethicin in channel formation studied using its covalent dimer N-terminally linked by disulfide bond.

Alamethicin is supposed to form helix-bundle-type channels by inserting the N terminus into bilayer lipid membranes under sufficient voltages. The N-terminal insertion has been studied with an alamethicin dimer (di-alm) N-terminally linked by a disulfide bond and by the asymmetric addition of dithiothreitol (DTT) and tetrathionate (TT) to the membrane. When di-alm was added to the cis-side membrane, it forms long-lasting channels with the lifetime tau of about 100 ms at cis-positive voltages. The lifetime was reduced to a few milliseconds by addition of DTT to the cis-side membrane, indicating that most of the channels were formed by the monomers (alm-SH) that resulted from the cleavage of the disulfide bond in di-alm. The succeeding addition of TT to the trans-side produced channels of tau=10-20 ms besides the channels of alm-SH. The results suggested that TT reacted with the N-terminal thiol group of alm-SH located at the trans-side of the membrane to alter the lifetime. The N-terminal insertion of alamethicin helices by voltage activation, therefore, was confirmed.

Alamethicin↗
PubMed1998

An antagonist of substance P N-terminal fragments, D-substance P(1-7), reveals that both nociceptive and antinociceptive effects are induced by substance P N-terminal activity during noxious chemical stimulation.

Substance P (SP) N-terminal fragments are known to alter nociception when injected intrathecally or when released in response to capsaicin. However, it is not known whether a sufficient concentration of SP N-terminal metabolites accumulate during noxious stimulation to modulate nociception. To test this, we examined the effect of the SP(1-7) antagonist, D-SP(1-7), injected intrathecally in mice, on two nociceptive assays that are differentially affected by exogenous SP(1-7): acetic acid-induced writhing that is inhibited and formalin-induced behaviors that are enhanced by SP(1-7). One nmol of D-SP(1-7) is sufficient to block the acute (30 min) antinociceptive effects of SP(1-7) on writhing. When injected alone at much higher doses (10-100 nmol), D-SP(1-7) inhibited writhing. In the formalin assay, SP(1-7) had no acute effect (30 min) on responses during Phase 1 at any dose tested, but D-SP(1-7) increased responses 5 min after injection of low (2-1000 pmol), but not high doses (10 and 100 nmol). Twenty-four hours after injection of SP(1-7), writhing was inhibited and formalin responses were increased. D-SP(1-7) prevented these effects of SP(1-7) but had no effect when injected alone, indicating that there is no tonic SP N-terminal activity in mice not exposed to noxious stimuli. Thus, acetic acid and formalin each induce endogenous SP N-terminal activity, respectively, producing a pro-nociceptive effect that is relatively insensitive to D-SP(1-7) and antinociception that is very sensitive to inhibition by D-SP(1-7).

Analysis of Variance↗
PubMed1999

Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides.

A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor. To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae. A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability. The mutated VPE was converted by the action of the VPE that had been purified from castor bean. Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE. In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide. Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.

Amino Acid Sequence↗
PubMed1996

Site-specific substitution of inosine at the terminal positions of a pre-mRNA intron: implications for the configuration of the terminal base interaction.

Genetic evidence in yeast has revealed that a non-Watson-Crick base-pairing interaction between terminal guanosine residues of the intron is required for the second step of pre-mRNA splicing. To explore the likely configuration of the interaction between the terminal guanosines of the intron, inosine was uniformly incorporated into an adenovirus pre-mRNA substrate (Ade) to replace guanosine residues. Splicing of the inosine-containing Ade pre-mRNA was completely inhibited. Psoralen cross-linking reveals that the association of U1 and U2 snRNPs with the intron was impaired. To eliminate the deleterious effects caused by complete inosine replacement, guanosine residues at the splice site(s) of the Ade pre-mRNA were substituted by inosine. Such pre-mRNA substrates were obtained by ligation of two or three RNA fragments; the 3' piece was primed with inosine or mono-phosphate inosine. Splicing of the Ade pre-mRNA containing an inosine residue at the 5' or the 3' splice site, or at both sites proceeds normally. Thus, the functions of the terminal guanosine residues of the intron in splicing can be replaced by inosine. This result supports the previous notion that an N1-carbonyl symmetric interaction likely occurs between the intron terminal residues during pre-mRNA splicing.

Base Composition↗
PubMed1993

Exploring the interface between the N- and C-terminal helices of cytochrome c by random mutagenesis within the C-terminal helix.

Buried within cytochrome c lies a highly-conserved helix-helix interface formed by the perpendicular packing of the C-terminal helix against the N-terminal helix. This interface involves a peg-in-hole interaction between Gly-6 and Leu-94 and an aromatic-aromatic interaction between Phe-10 and Tyr-97. To gain insight into protein design, we investigated the relationship between the sequence of the interface and the physiological function of yeast iso-1-cytochrome c. A library of mutants at positions 94 and 97 of the C-terminal helix was created to examine the effect of novel amino acid combinations. We isolated 45 of the 400 possible amino acid combinations, 32 of which result in a functional cytochrome c. Contrary to evolutionary conservation of the peg-in-hole and aromatic-aromatic interactions, we find that side-chain volume and conservation of aromatic residues do not play an essential role in determining function. Additionally, we find negatively-charged residues within the interface that result in a functional cytochrome c. Examination of the 45 missense mutants indicates that approximately 120 unique combinations are compatible with function. These results show that the interface is flexible. However, truncation of the C-terminal helix at position 94 abolishes function, suggesting that the interface is essential. The correlation observed between our library of mutants and the mutation matrix compiled by Gonnet et al. [Gonnet, G. H., Cohen, M. A., & Benner, S. A. (1992) Science 256, 1443-1445] demonstrates the potential use of the matrix to predict the effect of sequence changes on natural proteins and to optimize the design of novel proteins.

Amino Acid Sequence↗
PubMed1994

Self-association and DNA binding of lambda cI repressor N-terminal domains reveal linkage between sequence-specific binding and the C-terminal cooperativity domain.

The effects of temperature, protons, and KCl on self-assembly and site-specific binding of lambda cI N-terminal domains with operator sites OR were studied to assess the roles of these domains in DNA binding and cooperativity of the natural system. Domain self-assembly was studied using sedimentation equilibrium while domain-OR interactions were analyzed by quantitative DNase footprint titration. The self-assembly reactions were modeled best as a monomer-dimer-tetramer stoichiometry. Compared with intact cI, the monomer-dimer assembly is energetically weak and is largely independent of pH and KCl. The van't Hoff enthalpy of dimerization was found to be large and positive (+ 10.8 kcal/mol), in sharp contrast to that of intact cI (i.e., -16.1 kcal/mol; Koblan & Ackers, 1991a), indicating that different driving forces dominate the respective assembly processes. The interactions of OR with N-terminal domains were noncooperative under all conditions studied. Binding at each site is accompanied by a negative enthalpy (large at site 1, small at sites 2 and 3). Identical values for salt release and proton absorption were found for the three sites. Comparisons with the analogous thermodynamic parameters from our previous studies indicate that N-terminal domains exhibit different linkages to pH, KCl, and T from those of intact cI-OR interactions. This implies that the domains do not act independently within the intact repressor. Since the linkage differences are dependent upon which site the proteins are binding, the C-terminal domain must play a role in repressor discrimination between specific sites.

Allosteric Regulation↗
PubMed1999

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.

Amino Acid Sequence↗
PubMed2003

Does rapid atrial pacing terminate atrial fibrillation? A comparison of laboratory and device termination studies.

Atrial fibrillation is understood to be a re-entrant arrhythmia and for many years electrophysiologists have attempted to pace-terminate the rhythm. Several studies have demonstrated that an excitable gap is present during atrial fibrillation and that the capture of small amounts of atrial tissue is possible. Early attempts to terminate atrial fibrillation however were unsuccessful. The rapid development of pacemaker and defibrillator technology has provided an exciting new direction for the treatment of recurrent symptomatic atrial fibrillation. Results from studies of the effectiveness of atrial anti-tachycardia pacing algorithms have suggested that 50% of atrial arrhythmias (including atrial fibrillation) can be pace-terminated. These findings conflict with data from the electrophysiology laboratory where atrial fibrillation has yet to be convincingly terminated. In this review, the current literature is discussed and possible reasons for this discrepancy are proposed.

Algorithms↗