Surface plasmon resonance for measuring TBP-promoter interaction

A procedure was developed for real-time measurement of the interaction between an archaeal TATA-binding protein (TBP) with stress-gene promoters from the archaeon Methanosarcina mazeii using surface plasmon resonance (SPR), the BIACORE 3000 equipment, and the SA (streptavidin) Sensor Chip. Measurements were based on the SPR optical phenomenon, which resulted in light extinction when TBP bound a promoter. This process, detected as a change in a particular angle, was recorded in a sensorgram. The BIA-evaluation program allowed the calculation of the equilibrium constant (K(A)) of the interaction of M. mazeii TBP with the promoters of the stress genes grpE, hsp70(dnaK), and hsp40(dnaJ) (0.47, 0.26, and 1.21x10(7)M(-1), respectively) and, for comparison, with the promoter of a non-heat-shock gene, orf16 (0.08x10(7)M(-1)). The association rate (k(a)) of the non-heat-shock gene orf16 was 0.4x10(4)M(-1)s(-1) and those for the stress genes, grpE, hsp70(dnaK), and hsp40(dnaJ) were higher: 2.8, 1.5, and 3.5x10(4)M(-1)s(-1), respectively. The new procedure will allow a comparative analysis of different TPBs and promoters (wild type and mutants) under physiologic and stress conditions, and a correlation of TBP binding parameters with constitutive and stress-induced gene expression.     

Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal

We studied various aspects of heat‐shock response with special emphasis on the expression of heat‐shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5‐fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.     

Hsp70 Induction and hsp70 Gene polymorphisms as Indicators of acclimatization under hyperthermic conditions

The possibility of using Hsp70 and hsp70 gene polymorphisms as markers of acclimatization was investigated. Volunteers (22) were subjected to an acclimatization regimen and blood analysed for Hsp70 (Hsp72) and hsp70 polymorphisms before and after a heat tolerance test. Physiological parameters denoting acclimatization, or not, were correlated to levels of Hsp70 and combination of hsp70 genes. Only individuals that acclimatized had decreased basal Hsp70 levels and increased ability to induce Hsp70 together with a specific hsp70 genotype combination. We propose that Hsp70 levels (basal vs. induced) with the genotype combination have the potential to be used as markers of acclimatization.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

The genes coding for the hsp70 (dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

The hsp70(dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic bioconversions. The locus' genes and organization, 5'-grpE-hsp70(dnaK)-hsp40 (dnaJ)-trkA-3', are the same as those of the closely related mesophile Methanosarcina mazei S-6, but different from those of the only other archaeon for which comparable sequence data exist, the thermophile Methanobacterium thermoautotrophicum deltaH, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the deltaH proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion--by comparison with homologs from gram-negative bacteria first described in the S-6 molecule and later found to be present in all homologs from archaea and gram positives. The genes responded to a temperature elevation in a manner that demonstrated that they are heat-shock genes, functionally active in vivo. Ammonia also induced a heat-shock type of response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a typical heat-shock response.