Likewise, even when new Hsp64 was not being actively synthesized during recovery from HS (Fig 1A), the level of Hsp64, detected with the SPA805 antibody, remained unchanged (Fig 1B)

Likewise, even when new Hsp64 was not being actively synthesized during recovery from HS (Fig 1A), the level of Hsp64, detected with the SPA805 antibody, remained unchanged (Fig 1B). or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, Rabbit polyclonal to ATP5B the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3 untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the -galactosidase activity in 2 transgenic lines carrying the reporter gene under promoter and different lengths of the 5UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5UTR. INTRODUCTION A rapid induction of synthesis of a new set of polypeptides is the hallmark of the response mounted by nearly all cell types of most living organisms when subjected to thermal or several other cellular stresses (Ashburner 1982; Feder SRI-011381 hydrochloride and Hoffmann 1999). This rapidly induced SRI-011381 hydrochloride synthesis of the heat shock proteins (Hsps) is generally the result of transcriptional activation of heat shock (HS) genes, followed by a quick and preferential translation of the HS messenger ribonucleic acid (mRNA) (Morimoto et al 1994; Wu et al 1994, 1995). However, some exceptions to such a general paradigm for cellular response to thermal and other stresses have been reported earlier (Feder and Hoffmann 1999; Lakhotia 2001a, 2001b). Earlier studies in our laboratory (Lakhotia and Singh 1989, 1996; Singh and Lakhotia 1995) revealed that unlike in other tissues HS fails to induce any of the usual Hsps in Malpighian tubules (MTs) of larvae. Instead, synthesis of a different set of proteins, with a 64-kDa protein being the most prominent, is induced immediately after a 30-minute HS. Further studies (Lakhotia and Singh 1996) established that the HS-induced 64-kDa protein in the larval MT of was a member of the Hsp60 family. Krebs and Feder (1997) confirmed our observation on noninducibility SRI-011381 hydrochloride of the Hsp70 immediately after HS but showed that the Hsp70 did appear in the MT after some hours of recovery from HS and persisted till 21C24 hours. In several other insect species also, the HS response in MT has been reported to be somewhat different from that in most other cell types (Nath and Lakhotia 1989; Tiwari et al 1995, 1997; Singh and Lakhotia 2000). It appears therefore that the HS response in MT of insects is regulated in a manner different from that in other tissues. A recent study in our laboratory (Lakhotia and Prasanth 2002) on SRI-011381 hydrochloride transcription of the individual genes in response to HS in different cell types of revealed tissue- and developmental stageCspecific variations in induction and stability of the hsp70 transcripts. During the course of this study, we noted that the genes were SRI-011381 hydrochloride as quickly activated in response to HS in the larval MT cells as in other tissues, notwithstanding the fact that the Hsp70 did not appear till some time after recovery from the stress. In the present study, therefore, we examined the time kinetics of the induction of synthesis of Hsp70 and Hsp64 and their steady-state levels in larval MT cells immediately after HS and during recovery. Our results show that the regulation of synthesis and turnover of Hsp70 and Hsp64 during and after HS in MTs of larvae is.