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PubMed1991

The 20 kDa C-terminally truncated form of pertussis toxin subunit S1 secreted from Bacillus subtilis.

The subunit S1 of pertussis toxin (PT) was purified as the recombinant product BacS1 from the culture supernatant of a Bacillus subtilis strain containing a secretion vector with a DNA fragment coding for the mature subunit S1 inserted downstream of the signal sequence of the alpha-amylase gene. The method of purification was successive ion exchange and adsorption chromatography. BacS1 occurred in two forms (28 and 20 kDa) of which the truncated 20-kDa peptide was the main one in the supernatant. The truncated BacS1 was purified and shown to have the same NH2-terminus as the full-size (28 kDa) BacS1. It was also enzymatically active indicating correct conformation. The truncated BacS1 was also shown to elicit neutralizing and protective antibodies when injected into mice or rabbits.

Amino Acid Sequence↗
PubMed1990

Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum.

Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions. However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends. During the study of this phenomenon, we obtained KmR Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA. Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region. The most conspicuous result from Southern analyses on four such KmR plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence. Four truncated T-DNA insertions have been cloned. Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA. Another is missing the left T-DNA part and the last T-DNA is lacking both ends. Sequence analysis of the T-DNA::plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence. S1-type mapping of the most strongly expressed plant genome::nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic Pnos-nptII plants.

Base Sequence↗
PubMed2004

Regulation of HSF1-responsive gene expression by N-terminal truncated form of p73alpha.

DNp73 is a transactivation domain (TAD)-truncated form of p73. The ability of DNp73alpha to regulate gene expression was examined using reporter assays with luciferase gene constructs. Among various promoter-regulated reporter genes tested, heat shock factor (HSF)-responsive gene expression was selectively activated by DNp73alpha, but not by other p73-isoforms with TAD and DNp73beta. Deletion of TAD endowed p73alpha with the ability to activate HSF-responsive gene expression, but deletion of N-terminal proline-rich domain (PRD) rendered both DNp73alpha and the TAD-deleted p73alpha inactive. Considering the inability of DNp73beta, which is the C-terminus-truncated form of DNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DNp73alpha to be able to activate the HSF-dependent gene expression. In addition to the reporter gene expression, both DNp73alpha and TAD-deleted p73alpha activated the expression of an endogenous gene, hsp70, corresponding with an increase in the active form of HSF1. Taken together, these results demonstrate that TAD-truncated p73alpha can activate HSF-dependent gene expression via induction of active HSF1.

Base Sequence↗
PubMed2006

Over-expression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity.

In addition to regulating growth hormone release from the pituitary, ghrelin receptors also influence cell proliferation and apoptosis. By studying mitogen-activated protein kinase activity in human embryonic kidney 293 cells over-expressing ghrelin receptors, we aimed to identify the specific cell signalling pathways used by ghrelin receptors, and to determine if the truncated ghrelin receptor polypeptide had any influence on the functional activity of ghrelin receptors. We found that ghrelin activated extracellular signal-regulated kinases 1/2 with an EC50 value of 10 nM, and that this response was inhibited by the ghrelin receptor antagonists D-Lys3-GHRP-6 and [D-Arg1,D-Phe5,D-Trp(7,9),Leu11]-substance P. Ghrelin had little or no effect on the activity of c-Jun N-terminal kinase, p38 kinase or Akt. Ghrelin appeared to activate extracellular signal-regulated kinases 1/2 through a calcium-independent novel protein kinase C isoform which may utilize diacylglycerol derived from hydrolysis of phosphatidylcholine rather than from phosphatidylinositol. Ghrelin-stimulated extracellular signal-regulated kinases 1/2 activity was independent of transactivation of epidermal growth factor receptors, and even when ghrelin receptor internalization was blocked by concanavalin A or a beta-arrestin mutant, there was no decrease in phosphorylated extracellular signal-regulated kinases 1/2, suggesting this is a G protein-dependent process. The truncated ghrelin receptor polypeptide had no effect on ghrelin receptor signalling to extracellular signal-regulated kinases 1/2, but decreased the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors. In conclusion, our results suggest that any up-regulation of the truncated ghrelin receptor polypeptide might preferentially attenuate functional activity dependent on the constitutive activation of ghrelin receptors, while leaving ghrelin-dependent signalling unaffected.

Blotting, Western↗
PubMed2005

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein.

We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.

Base Sequence↗
PubMed2004

In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity.

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1-29) and a modified PTH(1-21) (MPTH(1-21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1-29), although active, was approximately 20-fold less potent than PTH(1-34) in a rat model of osteogenesis while MPTH(1-21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.

Animals↗
PubMed2006

Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats.

Human truncated tau protein is an active constituent of the neurofibrillary degeneration in sporadic Alzheimer's disease. We have shown that modified tau protein, when expressed as a transgene in rats, induced AD characteristic tau cascade consisting of tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau complexes. These pathological changes led to the functional impairment characterized by a variety of neurobehavioural symptoms. In the present study we have focused on the behavioural alterations induced by transgenic expression of human truncated tau. Transgenic rats underwent a battery of behavioural tests involving cognitive- and sensorimotor-dependent tasks accompanied with neurological assessment at the age of 4.5, 6 and 9 months. Behavioural examination of these rats showed altered spatial navigation in Morris water maze resulting in less time spent in target quadrant (p<0.05) and fewer crossings over previous platform position (p<0.05) during probe trial. Spontaneous locomotor activity and anxiety in open field was not influenced by transgene expression. However beam walking test revealed that transgenic rats developed progressive sensorimotor disturbances related to the age of tested animals. The disturbances were most pronounced at the age of 9 months (p<0.01). Neurological alterations indicating impaired reflex responses were other added features of behavioural phenotype of this novel transgenic rat. These results allow us to suggest that neurodegeneration, caused by the non-mutated human truncated tau derived from sporadic human AD, result in the neuronal dysfunction consequently leading to the progressive neurobehavioural impairment.

Alzheimer Disease↗
PubMed2004

A restricted maximum likelihood estimator for truncated height samples.

A restricted maximum likelihood (ML) estimator is presented and evaluated for use with truncated height samples. In the common situation of a small sample truncated at a point not far below the mean, the ordinary ML estimator suffers from high sampling variability. The restricted estimator imposes an a priori value on the standard deviation and freely estimates the mean, exploiting the known empirical stability of the former to obtain less variable estimates of the latter. Simulation results validate the conjecture that restricted ML behaves like restricted ordinary least squares (OLS), whose properties are well established on theoretical grounds. Both estimators display smaller sampling variability when constrained, whether the restrictions are correct or not. The bias induced by incorrect restrictions sets up a decision problem involving a bias-precision tradeoff, which can be evaluated using the mean squared error (MSE) criterion. Simulated MSEs suggest that restricted ML estimation offers important advantages when samples are small and truncation points are high, so long as the true standard deviation is within roughly 0.5 cm of the chosen value.

Anthropometry↗
PubMed2004

The P3 domain of E. coli ribonuclease P RNA can be truncated and replaced.

We prepared some truncated and replaced P3 mutants of Escherichia coli RNase P RNA, and used them to examine the RNase P ribozyme and holoenzyme reactions of a pre-tRNA substrate. The results indicated that mutations in the P3 domain did not affect the cleavage site selection of the pre-tRNA substrate, but did affect the efficiency of cleavage of the substrate. Results of stepwise truncation of the P3 domain and its replacement by the TAR sequence showed that the P3 domain of the E. coli RNase P was able to be truncated to certain length and was replaceable, but could not be deleted in the ribozyme.

Base Sequence↗
PubMed2005

C-terminal truncation modulates both nucleation and extension phases of tau fibrillization.

Proteolytic post-translational modification has been proposed as an early stage event in the aggregation of tau protein and formation of neurofibrillary lesions in Alzheimer's disease. Caspases and other proteases cleave tau in vivo at discrete locations including Asp421 and Glu391. Both cleavage products are prone to aggregation relative to wild-type, full-length tau protein. To determine the mechanism underlying this effect, the fibrillization of tau truncated after Asp421 and Glu391 residues was characterized in a full-length four-repeat tau background using quantitative electron microscopy methods under homogeneous nucleation conditions. Both C-terminal truncations decreased critical concentration relative to full-length tau, resulting in more filament mass at reaction plateau. Moreover, truncation directly augmented the efficiency of the nucleation reaction. The results suggest the mechanism through which C-terminal proteolysis can modulate tau filament accumulation depending on whether it precedes or follows nucleation.

Alzheimer Disease↗
PubMed2004

A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain.

Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.

Amino Acid Sequence↗
PubMed2004

Domain swapping in N-truncated human cystatin C.

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.

Cystatin C↗
PubMed2005

N-terminally truncated variant of the mouse GAIP/RGS19 lacks selectivity of full-length GAIP/RGS19 protein in regulating ORL1 receptor signaling.

The regulators of G protein signaling (RGS) are a family of proteins with conserved RGS domains and play essential roles in regulating G protein-mediated signal transduction and physiological events. GAIP/RGS19 (G alpha interacting protein, also classified as RGS19), a member of the RGS family, has been shown to negatively regulate the signaling of many G protein-coupled receptors, including the opioid receptors. Two GAIP/RGS19 mRNA variants, resulted from an alternative splicing of exon 2 of the GAIP/RGS19 gene, were identified in multiple mouse tissues. One of the transcripts consists of a complete set of exons and encodes a full-length GAIP/RGS19 protein, and the other does not have exon 2 and therefore encodes an N-terminal 22 residue truncated short GAIP/RGS19 protein. When co-expressed with either the opioid-receptor-like (ORL1) receptor or one of the mu, delta, and kappa opioid receptors, by transfecting dual-expression plasmids into COS-7 cells, the full-length GAIP/RGS19 was more effective than the N-terminally truncated variant and was more selective in regulating the ORL1 receptor signaling than in regulating the mu, delta, and kappa opioid receptors, as measured by the effectiveness to increase the agonist-stimulated GTPase activity and to reverse the agonist-induced inhibition of cyclic AMP accumulation. In the same assays, the N-terminally truncated GAIP/RGS19 did not distinguish ORL1 from the mu, delta, and kappa opioid receptors. In contrast, co-expression of RGS4 with either ORL1 or opioid receptors showed the selectivity of RGS4 for regulating opioid receptors was mu > kappa > delta > ORL1, an order completely different from that of GAIP/RGS19. The results suggest that GAIP/RGS19 prefers regulating ORL1 receptor signaling over other opioid receptors, and that the N-terminal domain of GAIP/RGS19 plays a crucial role in its receptor preference.

Alternative Splicing↗
PubMed2005

Development of a specific ELISA for the quantitative study of amino-terminally truncated beta-amyloid peptides.

Clinical studies for disease modifying drugs in Alzheimer's disease are in real need for a sensitive biochemical diagnostic and therapeutic marker. Encouraging results have been obtained by measuring levels of pathology related proteins such as amyloid beta (Abeta) peptides and tau proteins in cerebrospinal fluid (CSF) and plasma of patients. We and other research groups have shown that truncated Abeta11-40 and Abeta11-42 is also a potential marker and that it is produced and deposited in the brains of patients and transgenic mouse models. Because of a lack of quantitative methods for specific measurement of the truncated Abeta it has not been possible to evaluate the true value of this potential biomarker. To overcome these limitations we developed a novel monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to measure Abeta11-40 and Abeta11-42 levels in biological materials. For this purpose monoclonal antibodies were produced that react specifically with amyloid precursor protein (APP) products generated by cleavage at position 11 of the Abeta sequence. The assay has the sensitivity, selectivity and dynamic range to allow specific, direct quantitation of truncated Abeta11 peptides in cell culture medium, cerebrospinal fluid and brain tissue extracts.

Alzheimer Disease↗
PubMed2005

Relocation of truncated bid plays an important role in suppression of tumor necrosis factor alpha induced apoptosis in hepatocytes isolated from transgenic mouse.

BACKGROUND/AIMS: Tumor necrosis factor alpha (TNFalpha) induces apoptosis in murine hepatocytes pretreated with Actinomycin D (ActD) in vitro. This study sought to clarify the relationship between hepatic energy status and TNFalpha-induced hepatocyte apoptosis, using mice transgenic for creatine kinase (CK) expression. MATERIALS AND METHODS: Hepatocytes from CK transgenic mice were cultured with or without creatine (Cr). The concentrations of ATP and phosphocreatine (PCr) in hepatocytes were measured by high-pressure liquid chromatography. Sixteen hours after treatment with ActD and TNFalpha, we evaluated cell viability of these hepatocytes. We examined truncated Bid and cytochrome c by immunoblot analysis. RESULTS: Six hours after cell isolation, the concentration of PCr in CK transgenic hepatocytes cultured with Cr increased to 8.23 +/- 0.01 microg/mg protein, while that of hepatocytes cultured without Cr was lower than 0.1 microg/mg protein. In hepatocytes cultured without Cr, ActD and TNFalpha treatment induced massive cell death, while hepatocytes cultured with Cr maintained greater than 80% viability. In CK transgenic hepatocytes cultured with Cr, truncated Bid relocation to mitochondria was highly suppressed, compared to CK transgenic hepatocytes cultured without Cr. CONCLUSION: PCr accumulation may prevent TNFalpha-induced apoptosis in murine hepatocytes by suppression of truncated Bid targeting to mitochondria.

Animals↗
PubMed2003

Diverse regulation of full-length and truncated growth hormone receptor expression in 3T3-L1 adipocytes.

Two truncated forms of growth hormone (GH) receptor (GHR), 1-277 and 1-279, were reported to be normally produced in human tissues by alternative splicing in exon 9 and its boundary. We found previously that GHR-277 exerts a dominant-negative effect on full-length GHR (GHR-fl)-mediated GH signaling causing short stature. The existence of truncated GHRs (hGHR-tr) in normal tissues suggests that hGHR-tr may play a physiological role in regulation of GH action at the cellular level. To clarify the physiological significance of GHR-tr and the regulation mechanism of GHR-tr expression, we examined the expression of mouse GHR-tr (mGHR-tr) mRNA in mouse adipocyte 3T3-L1 cells, comparing with that of mouse GHR-fl (mGHR-fl). The mRNAs of two mGHR-tr, mGHR-282 and mGHR-280, were detected by RT-PCR methods using specific primers. Although the mGHR-282 and mGHR-280 mRNA levels were approximately 100 times lower than that of mGHR-fl in mature 3T3-L1 cells, quantitative analysis by competitive RT-PCR methods revealed that the mRNA levels of mGHR-280 in 3T3-L1 cells were transiently reduced and thereafter increased during differentiation from preadipocyte to adipocyte. In contrast, the mRNA levels of mGHR-fl were increased in parallel with the progress of differentiation. Stimulation by GH of differentiated 3T3-L1 mature adipocytes resulted in dose-dependent increases of the mRNA of both mGHR-fl and mGHR-282, whereas it caused a paradoxical decrease of the mRNA of mGHR-280 stimulated by high concentration of GH. These findings suggest that the expressions of truncated mGHRs were regulated in a different manner from that of mGHR-fl, thereby modulating GH action in murine adipocytes.

3T3 Cells↗
PubMed2005

C-terminal truncation affects kinetic properties of GluR1 receptors.

GluR1flop receptors in which the C-terminal 52 amino acids had been recombinantly removed were characterized with whole-cell recording and binding assays. Compared to wildtype GluR1, truncated receptors showed faster desensitization and deactivation and they recovered more slowly from desensitization. The EC50 for glutamate was increased 2-fold. In binding tests, K(D)s for [3H]fluorowillardiine were 1.5 times larger for truncated receptors. According to receptor simulations, most differences can be explained if the C-terminal domain is assumed to stabilize the ligand-bound closed and open states. The effects on response waveforms are different from those caused by phosphorylation, suggesting that the C-terminus influences receptor function in multiple ways. Truncated forms of GluR1 identical or similar to the one examined here may also be generated by calcium-activated proteases during intense synaptic activity. The lowered affinity and faster inactivation of these receptors suggests that their presence does not represent a risk for neuronal viability.

Amino Acid Sequence↗
PubMed2004

Transcription of subtelomerically located var gene variant in Plasmodium falciparum appears to require the truncation of an adjacent var gene.

The Plasmodium falciparum R29 clone preferentially transcribes the R29var gene variant on rosette selection, unlike other isogenic clones from the same parasite lineage. Characterisation of the R29var gene locus revealed that this gene lies internal to, and is in a tail-to-tail orientation with, a second var gene variant (A4var) at one end of chromosome 13. In the R29 clone, a spontaneous deletion event between these two var variants deletes all of the A4var gene and the subtelomeric repetitive sequence arrays. We have previously shown that a simple disruption of the A4var gene is not sufficient to preferentially activate the R29var gene in rosette-selected parasites. We therefore hypothesised that the truncation of the chromosome end may be a key factor in predisposing the R29var variant to transcription under rosette selection conditions. Here, we have generated a panel of isogenic parasite clones with both intact and truncated A4var-R29var loci, and show that R29var transcription is only detected in rosette-selected clones with a truncated locus. Furthermore, we present provisional data describing the relative frequency with which this spontaneous deletion event occurs. These data have implications in our understanding of how spontaneous deletion events within subtelomeric var loci may affect transcription of these var gene variants.

Animals↗