can overcome this inhibition in vitro. However, while this ob- servation does provide one possible explanation for the ability of IE2 to counter Dr1-mediated repression of the hsp70 pro- moter in vivo and is consistent with the known TATA depen- dency of IE2, it should be noted that other mechanisms may also be involved. Firstly, it is possible that nonphosphorylated forms of Dr1 act at multiple steps, as it has been suggested that rDr1 inhibits transcription by preventing the entry of TFIIB into the preinitiation complex (25). The experiments presented here do not address this activity of rDr1, nor do they rule out the possibility that IE2 also acts at this point. Secondly, both forms of Dr1, the phosphorylated hDr1 and the rDr1-like TFIID-associated form, are present during in vivo assays; while our results indicate that IE2 is not able to disrupt the interac- tion of hDr1 with DNA-bound TBP in vitro, we cannot rule out that IE2-mediated derepression of the hsp70 promoter in vivo may occur by indirect alleviation of this or other as-yet-un- known activities of hDr1. To rule out this latter possibility requires greater understanding of the relative contributions of both forms of cellular Dr1 to transcriptional repression and, in turn, of how the various activities of Dr1 are regulated by phosphorylation within a cell. Finally, it is likely that the ability of IE2 to alleviate Dr1-mediated repression is simply one of a number of mechanisms by which IE2 can activate target pro- moters. Current understanding of the nature of IE2 indicates that it is a versatile protein, capable of interacting with several cellular transcription factors and up-regulating gene expres- sion by diverse mechanisms. Although it is difficult at present to quantify the contributions of these various effects to trans- activation by IE2 as a whole (a problem which is likely to be exacerbated if more than one mechanism operates at the same promoter), these studies provide further insight into the role of IE2 as a multifunctional transactivator protein.
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contains multiple binding sites for the sequence-specific regulatory proteins, GAGA factor (GAF) and heat-shock factor (HSF). Prior to heat shock, GAF resides on the Hsp70 promoter (G ilmour et al. 1989; O’B rien et al. 1995). The binding of GAF appears to maintain the promoter region in a nucleosome-free conformation (K arpov et al. 1984; U dvardy et al. 1985; N acheva et al. 1989; B ecker and C raig 1994; T sukiyama et al. 1994; G eorgel 2005), as is consistent with the DNase hyper- sensitivity of the region (W eber et al. 1997). The poly- merase apparatus is preassembled but stalled, awaiting the arrival of activated HSF. Thus, the Hsp70 promoter is ‘‘bookmarked’’ (X ing et al. 2005). These features are thought to allow ready access of the general transcrip- tion factors to the core promoter and thus facilitate rapid induction of transcription upon heat shock, but may thereby facilitate the access of the transposition machinery to the underlying DNA, especially at or near the DNase hypersensitive sites. Finally, although nomi- nally a heat-shock gene, Hsp70 is transcribed in the male germline (B outanaev et al. 2002; L akhotia and P rasanth 2002), which should to facilitate germline transposition. Indeed, numerous transposons have naturally and independently inserted into the Hsp70 proximal promoter (M ichalak et al. 2001; Z atsepina et al. 2001; B ettencourt et al. 2002; L erman et al. 2003; L erman and F eder 2005; our unpublished data).
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Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry ⫹ , ⌬2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry ⫹ , ⌬2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhance- ment of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA.
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ABSTRACT Controlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila developmental genetics for nearly 30 years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special upstream activation sequence (UAS) promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a signiﬁcant technical limitation. Here, we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed Piwi-interacting RNAs (piRNAs) generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.
Figure 1.—Constructs and transgenic lines. Drawings represent the I factor (top) and the different transgenes used in this study. ORF1 and ORF2 are indicated as open boxes while untranslated re- gions from the I factor (5⬘I and 3⬘I, 5⬘ and 3⬘ untrans- lated regions, respectively) are indicated as solid boxes. Light strippled boxes indicate the promoter (phs) and the terminator region (ths) of the hsp70 gene, while heavy strippled boxes indicate the 3⬘ end of the P element (3⬘P). Arrows indicate the position of transcription ini- tiation from the I factor or the hsp70 promoter. Parts of the transgenes that are irrelevant to the study (white sequences and the 5⬘ end of the P element) are omitted. On the left of the drawings are the names of the different transgenes. On the right of the drawings are the names of the transgenic lines derived from the wK and JA strains, and the last column summarizes the repressor effects of the transgenes on I factor activity.
Expression of CSP in M. smegmatis and M. bovis BCG. A gene encoding P. falciparum CSP was introduced into M. smeg- matis and M. bovis BCG to examine their ability to express foreign DNA. The gene encoding the 42-kDa antigen P. falci- parum CSP was chosen because it is a well-characterized target of the immune response in persons with malaria (7). The CSP DNA, which was amplified by PCR and identified by sequenc- ing, was inserted into the unique BamHI and KpnI sites of Mycobacterium-E. coli shuttle vector pBCG2100 containing the HSP70 promoter to create pBCG/CSP. M. smegmatis and M. bovis BCG were transformed with plasmid pBCG/CSP, and
Effects of metalloproteinase expressed in B. mori larvae. A recombinant BmNPV BmMMP, carrying XcGV mmp under the polyhedrin promoter, was constructed to purify and char- acterize the putative metalloproteinase. The endogenous cys- teine proteinase gene of BmMMP was disrupted by insertion of a Drosophila hsp70 promoter-lacZ gene cassette generating BmMMPCysPD in order to avoid any effect of the cysteine proteinase of BmNPV (26). BmMMPCysPD was purified by a plaque assay (identified by blue plaques), and the hsp70-lacZ insertion was confirmed by PCR using virus genomic DNA as a template. PCR amplification of WT BmNPV DNA using primers Cyspro1 and Cyspro2 produced a 1-kb product. The PCR product amplified from the corresponding regions of BmMMPCysPD and BmCysPD containing the 3.5-kb hsp-lacZ cassette was about 4.5 kb, which indicates that the cysteine proteinase was disrupted (data not shown).
dissociation from peptide-charged Hsp70 molecules, using an Hsp110-like nucleotide exchange mechanism. Homodimer formation might have enabled tight coordination between Hsp70 molecules during collaborative folding of larger, possibly multi-domain protein substrates. Specifically, a peptide-charged Hsp70 molecule might have recruited an ATP- bound Hsp70 molecule, sensing its nucleotide-dependent conformation. While nucleotide exchange would have resulted in substrate dissociation from the same Hsp70 molecule, unmasked hydrophobic peptide segments could have efficiently been rebound by the adjacent accessible peptide binding cleft of the second Hsp70 molecule. Thus, two Hsp70 molecules would have been coupled in the hypothetical homodimeric Hsp70 complex similar to a two- stroke engine. Intriguingly, most key residues mediating the interactions between Sse1p and Hsp70 are conserved among sequences of canonical Hsp70s. However, using our MABA- ADP dissociation assay, we were not able to find evidence for an NEF function of canonical Hsp70s (data not shown). Possibly, present canonical Hsp70s have lost this function after the emergence of specialized NEFs and the concomitant relief of evolutionary restraints. Hsp110/Grp170 family proteins appear to have specialized for their nucleotide exchange function: The systematic substitution of the 3HBD residue 554 from Ala in canonical Hsp70s to Glu in Hsp110s appears to reinforce the interaction between the 3HBD and the NBD, which is crucial for nucleotide exchange. Glu554 forms a salt bridge to Arg47 of the NBD. Furthermore, residues implicated in ATP hydrolysis and conformational cycling in canonical Hsp70s – both features which are dispensable for Hsp110 function – are less conserved in Hsp110 sequences. Moreover, residues which might promote unproductive homodimeric association (e.g. Ala300, human Hsp70 numbering) differ from canonical Hsp70 sequences.
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Following Proteinase K-agarose or control agarose treatments the HSP70 preparations were used to stimu- late macrophages. The cell-stimulating capacity of the HSP70 sample treated with Proteinase K-agarose beads was almost completely ablated relative to the HSP70 subjected to control agarose (Figure 2C). We also observed that treatment of HSP70 with the control agar- ose beads alone caused a substantial reduction in the level of endotoxin contamination of the HSP70 prepara- tion compared to the buffer-alone control sample (Fig- ure 2A). We speculate that the agarose beads may bind nonspecifically to endotoxin. Nevertheless, the endo- toxin content of HSP70 treated with Proteinase K-agar- ose or control agarose beads were not dramatically different, with an even greater amount of endotoxin observed in the HSP70 subjected to Proteinase K-agar- ose compared to the amount of endotoxin observed in the HSP70 subjected to control agarose (Figure 2A). These results strongly implicate a role for the protein component in the stimulatory effect of these recombi- nant HSP70 preparations. Moreover, they have allowed us to speculate that protein structural motifs found only in fully intact HSP70 are required for TLR4 activation, be it through direct interaction with the TLR4 receptor complex, or through its ability to facilitate the transfer of bound endotoxin to MD2 on the TLR4 receptor com- plex, or through a mechanism that involves sensitization of the TLR4 receptor complex to levels of endotoxin that would normally be below levels required to induce robust signaling.
P]UTP and T7 RNA polymerase according to instructions provided by the manufacturer (Takara, Dalian, China). The contaminating cDNA templates were removed through DNase digestion according to instructions provided by the manufacturer (NEB Co., Beverly, MA). RNA transcripts were then further purified using G-50 Nick columns according to the manufacturer’s instructions (GE Healthcare, Bucks, United Kingdom). The quality of each RNA transcript was determined using sequencing gels. RNA transcripts prepared from a construct carrying a partial PVX sequence (nucleotides 3183 to 3457) were used as a control for the assay. To prepare radioactive single-stranded RNA, the PCR products were digested with BamHI to remove one T7 promoter prior to in vitro transcription. Long double-stranded or single-stranded GFP RNA (268 bp) was transcribed from the PCR fragments amplified with primers GFP-T7-F and GFP-T7-R or GFP-F and GFP-T7-R (Table 1). The resulting radioactive RNA transcripts were mixed with purified His 6 -tagged NSvc4 in 13.5 l binding buffer containing
10. Mukhopadhyay I, Saxena DK, Chowdhuri DK. Hazardous effects of effluent from the chrome plating industry: 70kDa heat shock protein expression as a marker of cellular damage in transgenic Drosophila melanogaster (hsp70 lac Z). Environ Health Perspec 2003; 3: 1926- 1932.
the ERM must be related with dento-alveolar ankylosis . Furthermore, Yamashiro et al. reported that denerv- ation of the inferior alveolar nerve leads to a reduced distribution of the ERM at 1 week and dento-alveolar ankylosis occurs at 6 weeks. They also confirmed that ERM receptor was immune-positive for trkA, which shows a high-affinity to the NGF receptor and they con- cluded that the sensory nerve might play a regulatory role in maintaining the ERM . Mine et al. observed the healing process of the PDL after tooth re-plantation in rats and compared the occluded group with the non- occluded group. They found that dento-alveolar anky- losis was clearly detected in the non-occluded group, but was not detected in the occluded group. They con- cluded that occlusal stimuli promote the regeneration of the PDL and prevent dento-alveolar ankylosis . These facts suggest that the reduction of ERM distribution in the PDL might precede the development of dento- alveolar ankylosis and that mechanical stimuli must prevent dento-alveolar ankylosis. However, only a few studies have been reported about changes of the ERM under the various kinds of mechanical forces in vitro . In general, the stress response is considered to repre- sent a cellular defense mechanism against environmental disturbances [9–12]. If cells are exposed either to a mild or a moderate stress that is sufficient to up regulate the expression of heat shock proteins (HSPs), they are often able to survive subsequent, otherwise lethal stress stim- uli. HSPs can be expressed by all types of cells and they play a protective role against a variety of harmful factors, including oxidants, inflammation, hypoxia, hyperthermia and also mechanical stimuli include orthodontic force [10–14]. Furthermore, strong orthodontic force induced apoptosis of PDL fibroblasts, and many ERM cells also fell into apoptosis [15, 16]. It was confirmed that HSP70 which serves in maintaining homeostasis was acted as a cochaperone by HSP40, can inhibit apoptosis by interfer- ing with the function of apoptosis inducing factor (AIF) . The effect of HSP70 on apoptotic protease-activating facor 1 (Apaf-1) probably accounts for its ability to pro- vide resistance to the reported stress-induced apoptosis and its expression exists in the PDL throughout life .
Expression and purification of recombinant proteins To determine whether fusion of Mage-a1 to Hsp70 can in- crease the potency of vaccination against Mage-expressing tumors, we expressed Mus musculus Mage-a1, Hsp70, and Mage-a1-Hsp70 fusion proteins using the prokaryotic ex- pression vector pGEX4T1, which produces GST-tagged recombinant proteins (Figure 1A). Only part of the Mage- a1 coding sequence (aa 118–219) was cloned because full length Mage-a1 failed to produce detectable protein after IPTG induction. High level protein expression was in- duced by IPTG for the control pGEX4T1 vector and the three GST tagged vectors in E. coli Rosetta 2 (DE3) cells, and the proteins were purified by GST affinity chromatog- raphy (Figure 1B-D). A single predominate band of ex- pected size of the purified protein was isolated for each of these proteins, though additional faint bands could be ob- served in each purification, indicating that some level of
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compartments, and targeting of misfolded or abnormal proteins for degradation. Many aspects of protein–protein interactions are also specifically regulated by chaperones. The accumulation of unfolded proteins in the endoplasmic reticulum lumen can trigger the unfolded protein response, which is implicated in the shutdown of protein synthesis that is a hallmark of the response to ischemia and other severe cellular stresses (Paschen, 2003). Thus regulation of the state of protein folding and protein association is a central aspect of normal cellular homeostatsis, which is severely perturbed by ischemia and reperfusion. Despite a large number of studies demonstrating neuroprotection by the chaperone Hsp70, in both animal stroke studies (Plumier et al., 1997; Rajdev et al., 2000; Yenari et al., 1998, 1999) and cell culture models of ischemia (Papadopoulos et al., 1996; Xu and Giffard, 1997), the mechanism, or more likely mechanisms, of protection are poorly understood.
Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the serum concentrations of HSP70 (DYC1663E, Minneapolis, MN, USA), follicle stimulating hormone (FSH)(CSB-E06869r, Cusabio Biotech CO.,Ltd., Wuhan, China), luteinizing hormone (LH) (CSB- E12654r, Cusabio Biotech CO.,Ltd., Wuhan, China), es- tradiol (E2) (CSB-E05110r, Cusabio Biotech CO.,Ltd., Wuhan, China), progesterone (P) (CSB-E07282r, Cusabio Biotech CO.,Ltd., Wuhan, China), testosterone (T)(E- EL-0072c, Elabscience Biotechnology Co.,Ltd., Wuhan, China), insulin (CSB-E05070r, Elabscience Biotechnology Co.,Ltd., Wuhan, China), C-reactive protein (CRP) (E- EL-R0022c, Elabscience Biotechnology Co.,Ltd., Wuhan, China), interleukin (IL)-6(E-EL-R0015c, Elabscience Biotechnology Co.,Ltd., Wuhan, China), IL-18(E-EL- R0567c, Elabscience Biotechnology Co.,Ltd., Wuhan, China), tumor necrosis factor (TNF)-α. (E-EL-R0019c, Elabscience Biotechnology Co.,Ltd., Wuhan, China). There were standard curves used to calculate for all these kits. The coefficients of variation within and be- tween plates were less than 10%.
The associations between anti-HSP70 antibodies and AF type as well as HSP70 and anti-HSP70 antibody increases and AF recurrence after ablation may point to parallel phenomena or imply causality which remains to be clarified although spontaneous variation can not be ruled out with certainty since repeat measurements in the control group were not available. In that respect, our study should be viewed as hypothesis generating and more mechanistic work is necessary to explore the evolving field of autoimmunology in AF. This area of in- vestigation seems of particular clinical interest for AF prevention as HSP function may be induced and auto- immune responses suppressed .
The importance of HSP70 in the function and integ- rity of mitotic centrosomes and in mitosis progression has long been recognized. For example, Sse1, a member of the yeast HSP70 family, was reported to modulate the oligomeric state of the MT motor Cin8 to regulate the length of mitotic spindle . Furthermore, NEK6 phosphorylates HSP70 and targets it to the mitotic spin- dle to facilitate chTOG–TACC3 complex recruitment, thus promoting kinetochore microtubule stability . Overexpressed HSP70 was also shown to repair the heat shock-induced defects in the mitotic centrosome and facilitate subsequent progression through mitosis . Additionally, HSP70 was shown to accumulate at the cen- trosomes of the heat shocked cells and prevent the pro- teasomal degradation of centrosome proteins . These studies all suggested the possibility that HSP70 may regu- late centrosome functions to support the mitotic progres- sion, and we previously confirmed that HSP70 localizes to the mitotic spindle pole and is required for centro- some integrity, MT nucleation and spindle assembly . However, the question of how HSP70 regulates the func- tions of the mitotic centrosome remained unanswered. In this study, we utilized the enhanced resolution of 3D-SIM and GSD microscopy to demonstrate that HSP70 colocal- izes with PCM components, including PCNT, CEP215 and γ-tubulin. Loss of HSP70 significantly reduces the recruitment of these PCM components and disrupts their 3D assembly at the mitotic centrosome, leading to decreased MT nucleation. Since the proper functioning of the mitotic centrosome requires PCM components to be recruited in a highly organized manner, our results reveal that HSP70 permits MT nucleation and bipo- lar spindle assembly by promoting the recruitment and accurate 3D assembly of PCM components at the mitotic centrosome.
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HSPs are involved in several important cellular processes. They bind unfolded proteins and can either mediate correct folding and compartmentalisation in the case o f normal and functional proteins (Haiti, 1996; Bukau and Horwich, 1998) or proteasomal degradation of defective or unwanted proteins (Straus et a l, 1988; Wagner et a l, 1994; Bercovich et a l, 1997; Savel'ev et a l, 1998). The latter could include normal proteins that are destined for deactivation, or abnormal proteins that are mislocalized or defective due to misfolding or chemical modification, and whose accumulation could be toxic (Bush et a l, 1997). These chaperone functions are mediated by interaction with co-factors, by modulating chaperone- protein binding, or by mediating targeting to specific proteins and sub-cellular compartments (Frydman and Hohfeld, 1997). For example, the ubiquitin domain protein BAG-1 and CHIP ubiquitin ligase act cooperatively to shift the activity of the Hsp70 chaperone system to degradation (Hohfeld, 1998; Luders et a l, 2000; Demand et a l, 2001 ; Murata et a l, 2001). An Hsp70 co-factor, Hsp40 has also been shown to be involved in degradation o f proteins by the UPS (Lee et a l, 1996; Huang et a l, 2001). A functional switch between degradation and protein folding may be an important quality control process in the cell. The formation of aggresomes may also be a mechanism for the safe compartmentalisation o f misfolded or toxic proteins, and Hsp40 and Hsp70 have been shown to be recruited as part o f aggresome formation (Ryan et a l, 2002). Therefore HSP activation is beneficial to cells under
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would otherwise compromise brain health in various conditions of inflammation. Induced or transgenic overexpression of Hsp70 correlate to a significant reduction in brain infarct volume following ischemic insult (41). The exact mechanism of this antagonistic interaction, where an overexpression of Hsp70 is noted with a concurrent down regulation of iNOS, is not entirely clear. Yenari et.al, implemented a transient MCAO stroke model in transgenic mice exhibiting Hsp70 overexpression, through which the preliminary data illustrated increased Hsp70 association with NF-κB and IκB during reperfusion and recovery (79, 80). This increased association then prevents phosphorylation of IκB by IKK and consequently establishes the inhibition of NF-κB (51, 82). In doing so, Hsp70 overexpression illustrated to regulate the inhibition of NF-κB and subsequent downstream inflammatory components. Data from Yenari et.al allude to an Hsp70 mediated anti-inflammatory regulation and an inhibition of iNOS, which would warrant further characterization of Hsp70 and iNOS in disease conditions that are threatened by inflammatory consequences (80, 82).
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Regarding the 3 ’ UTR of HSP70 genes, two types of sequences were cloned and sequenced (GenBank JF449364 and JF449365), showing a high sequence diver- gence each other. After sequence comparison, they could be assigned to the two types of 3 ’ UTR (I and II) described in HSP70 genes of other Leishmania species (Figures 3 and 4). Thus, we determined that the 3’ UTR type I is 936 nucleotides long, and would correspond to the LbrM28_v2.2990 entry (and possibly other genes of the HSP70 gene cluster, see below), and the 3’ UTR type II is 932 nucleotides long and would be associated with LbrM28_v2.2970 entry. The 3’ UTR-II was found 100% identical with the genomic sequence located down- stream of LbrM28_v2.2970, whereas the 3 ’ UTR-I had 99.1% of sequence identity with the sequence located downstream of LbrM28_v2.2990 entry, suggesting that the cloned 3 ’ UTR-I might correspond to other HSP70 gene in the cluster (see below). According to the 3 ’ UTR-I sequence, the polyadenylation in the LbrM28_v2.2990 transcript would occur after two ade- nines, and 187 bp upstream of the putative polypyrimi- dine tract previously mentioned. Regarding the LbrM28_v2.2970 gene, the polyadenylation would take place, after an A-rich region of 11 residues, and 154 bp upstream of a C-rich polypyrimidine tract of 14 nucleo- tides in length and U/C ratio of 0.08 (5’CCCCC CCCCTCCCC 3’). It is a common feature that the pre- sence of adenosine residue precedes the polyadenylation sites of a large number of trypanosome mRNAs . It is believed that poly(A) polymerases prefer an initial adenosine residue for attachment of the poly(A) tail, and the selection of the polyadenylation site would be strengthened by the presence of adenosine residues . After determining the extent of the UTRs, it was pos- sible to define the intergenic region (IR) within the L.
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