Low-dose metronomic cisplatin as an antiangiogenic and anti-inflammatory strategy for cancer.

BACKGROUND: Conventional chemotherapy is based on the maximum tolerated dose (MTD) and requires treatment-free intervals to restore normal host cells. MTD chemotherapy may induce angiogenesis or immunosuppressive cell infiltration during treatment-free intervals. Low-dose metronomic (LDM) chemotherapy is defined as frequent administration at lower doses and causes less inflammatory change, whereas MTD chemotherapy induces an inflammatory change. Although several LDM regimens have been applied, LDM cisplatin (CDDP) has been rarely reported. This study addressed the efficacy of LDM CDDP on tumour endothelial cell phenotypic alteration compared to MTD CDDP. METHODS: Tumour growth and metastasis were assessed in bladder cancer-bearing mice treated with LDM or MTD gemcitabine (GEM) and CDDP. To elucidate the therapeutic effects of LDM CDDP, the change of tumour vasculature, tumour-infiltrating immune cells and inflammatory changes were evaluated by histological analysis and mRNA expression in tumour tissues. RESULTS: Tumour growth and bone metastasis were more suppressed by LDM CDDP + MTD GEM treatment than MTD CDDP + MTD GEM. Myeloid-derived suppressor cell accumulation was reduced by LDM CDDP, whereas inflammatory change was induced in the tumour microenvironment by MTD CDDP. CONCLUSION: LDM CDDP does not cause inflammatory change unlike MTD CDDP, suggesting that it is a promising strategy in chemotherapy.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.

Associations Between Recipients' Feelings of Guilt for Donor and Depressive Symptoms Before Living Kidney Transplantation.

BACKGROUND: Recipient depression before kidney transplantation needs to be treated to reduce poor posttransplant outcomes. For recipients who receive living kidney transplantation, feelings of guilt for potential donors may be factors related to the presence of depressive symptoms. This study aimed to examine the association between recipients' feelings of guilt for the donor and depressive symptoms before living kidney transplantation. METHODS: Participants included 178 patients in Sapporo City General Hospital, Hokkaido, Japan, who completed a questionnaire before having living kidney transplantation from April 2009 to May 2016. Feelings of guilt for the donor, depressive symptoms using the Beck Depression Inventory-II (BDI-II), relationship to the donor, dialysis period, and socio-demographic characteristics were assessed via a questionnaire. Multivariate regression analyses were performed to examine the association between feelings of guilt for the donor and BDI-II score after multiple imputations. RESULTS: The results showed that feelings of guilt for donors were associated with depressive symptoms, especially cognitive factors. CONCLUSIONS: These findings indicate that medical staff needs to address recipients' feelings of guilt for donors before living kidney transplantation.

Development of a rational framework for the therapeutic efficacy of fecal microbiota transplantation for calf diarrhea treatment.

BACKGROUND: Establishing fecal microbiota transplantation (FMT) to prevent multifactorial diarrhea in calves is challenging because of the differences in farm management practices, the lack of optimal donors, and recipient selection. In this study, the underlying factors of successful and unsuccessful FMT treatment cases are elucidated, and the potential markers for predicting successful FMT are identified using fecal metagenomics via 16S rRNA gene sequencing, fecal metabolomics via capillary electrophoresis time-of-flight mass spectrometry, and machine learning approaches. RESULTS: Specifically, 20 FMT treatment cases, in which feces from healthy donors were intrarectally transferred into recipient diarrheal calves, were conducted with a success rate of 70%. Selenomonas was identified as a microorganism genus that showed significant donor-recipient compatibility in successful FMT treatments. A strong positive correlation between the microbiome and metabolome data, which is a prerequisite factor for FMT success, was confirmed by Procrustes analysis in successful FMT (r = 0.7439, P = 0.0001). Additionally, weighted gene correlation network analysis confirmed the positively or negatively correlated pairs of bacterial taxa (family Veillonellaceae) and metabolomic features (i.e., amino acids and short-chain fatty acids) responsible for FMT success. Further analysis aimed at establishing criteria for donor selection identified the genus Sporobacter as a potential biomarker in successful donor selection. Low levels of metabolites, such as glycerol 3-phosphate, dihydroxyacetone phosphate, and isoamylamine, in the donor or recipients prior to FMT, are predicted to facilitate FMT. CONCLUSIONS: Overall, we provide the first substantial evidence of the factors related to FMT success or failure; these findings could improve the design of future microbial therapeutics for treating diarrhea in calves. Video abstract.

Chemotherapy-Induced IL8 Upregulates MDR1/ABCB1 in Tumor Blood Vessels and Results in Unfavorable Outcome.

Tumor endothelial cells (TEC) lining tumor blood vessels actively contribute to tumor progression and metastasis. In addition to tumor cells, TEC may develop drug resistance during cancer treatment, allowing the tumor cells to survive chemotherapy and metastasize. We previously reported that TECs resist paclitaxel treatment via upregulation of ABCB1. However, whether TEC phenotypes are altered by anticancer drugs remains to be clarified. Here, we show that ABCB1 expression increases after chemotherapy in urothelial carcinoma cases. The ratio of ABCB1-positive TEC before and after first-line chemotherapy in urothelial carcinoma tissues (n = 66) was analyzed by ABCB1 and CD31 immunostaining. In 42 cases (64%), this ratio increased after first-line chemotherapy. Chemotherapy elevated ABCB1 expression in endothelial cells by increasing tumor IL8 secretion. In clinical cases, ABCB1 expression in TEC correlated with IL8 expression in tumor cells after first-line chemotherapy, leading to poor prognosis. In vivo, the ABCB1 inhibitor combined with paclitaxel reduced tumor growth and metastasis compared with paclitaxel alone. Chemotherapy is suggested to cause inflammatory changes in tumors, inducing ABCB1 expression in TEC and conferring drug resistance. Overall, these findings indicate that TEC can survive during chemotherapy and provide a gateway for cancer metastasis. Targeting ABCB1 in TEC represents a novel strategy to overcome cancer drug resistance. SIGNIFICANCE: These findings show that inhibition of ABCB1 in tumor endothelial cells may improve clinical outcome, where ABCB1 expression contributes to drug resistance and metastasis following first-line chemotherapy.

Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer.

Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.

Accumulation of Laforin and Other Related Proteins in Canine Lafora Disease With EPM2B Repeat Expansion.

Canine Lafora disease (LD) is an autosomal recessive genetic disorder causing nonfatal structural epilepsy, mainly affecting miniature wirehaired dachshunds. Repeat expansion in the EPM2B gene causes a functional impairment of the ubiquitin ligase malin which regulates glycogen metabolism. Abnormally structured glycogen accumulates and develop polyglucosan bodies predominantly in the central nervous system. The authors performed a comprehensive clinical, genetic, and pathological study of 4 LD cases affecting miniature wirehaired dachshund dogs with EPM2B repeat expansions, with systemic distribution of polyglucosan bodies and accumulation of laforin and other functionally associated proteins in the polyglucosan bodies. Myoclonic seizures first appeared at 7-9 years of age, and the dogs died at 14-16 years of age. Immunohistochemistry for calbindin revealed that the polyglucosan bodies were located in the cell bodies and dendritic processes of Purkinje cells. Polyglucosan bodies were also positive for laforin, hsp70, α/ß-synuclein, ubiquitin, LC3, and p62. Laforin-positive polyglucosan bodies were located in neurofilament-positive neurons but not in GFAP-positive astrocytes. In nonneural tissues, periodic acid-Schiff (PAS)-positive polyglucosan bodies were observed in the heart, skeletal muscle, liver, apocrine sweat gland, and smooth muscle layer of the urinary bladder. In the skeletal muscle, polyglucosan bodies were observed only in type 1 fibers and not in type 2 fibers. The results indicate that although the repeat expansion of the EPM2B gene is specific to dogs, the immunohistochemical properties of polyglucosan body in canine LD are comparable to human LD. However, important phenotypic variations exist between the 2 species including the affected skeletal muscle fiber type.

Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR.

To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551-9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly.

Tumor endothelial cells express high pentraxin 3 levels.

It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract.

Melissococcus plutonius is an important pathogen that causes European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M. plutonius strains that are phenotypically and genetically distinct from other strains. These strains belong to clonal complex (CC) 12, as determined by multilocus sequence typing analysis, and show atypical cultural and biochemical characteristics in vitro compared with strains of other CCs tested. Although EFB is considered to be a purely intestinal infection according to early studies, it is unknown whether the recently found CC12 strains cause EFB by the same pathomechanism. In this study, to obtain a better understanding of EFB, we infected European honeybee (Apis mellifera) larvae per os with a well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically. Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells degenerated, and some bacterial cells were detected outside of the midgut. However, they did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM was stained with anti-M. plutonius serum in most of the DAT561-infected larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the inside. These results suggest that infection of CC12 strain in honeybee larvae is essentially confined to the intestine. Moreover, our results imply the presence of M. plutonius-derived substances diffusing into the larval tissues in the course of infection.

Microminipigs as a new experimental animal model for toxicological studies: comparative pharmacokinetics of perfluoroalkyl acids.

In this study, we evaluated the efficacy of a novel minipig strain, the Microminipig (MMPig), as an animal model for studying the pharmacokinetics of a mixture of 10 perfluoroalkyl acids (PFAAs). After a single oral dose was given, we found that the blood depuration of PFAAs (blood t1/2), which we calculated using first-order elimination curves, ranged from 1.6 to 86.6 days. Among the five body compartments analyzed, the liver was the greatest site of accumulation of perfluorooctanesulfonate and longer chain perfluorinated carboxylates such as perfluorodecanoic acid, perfluoroundecanoic acid and perfluorododecanoic acid. We observed an increasing accumulation trend of perfluorinated carboxylates in the organs associated with the fluorinated carbon chain length. The perfluorononanoic acid burden was the highest among the treated compounds 21 days after a single exposure, as 29% of the given perfluorononanoic acid dose was accumulated in the tissues. The persistence of PFAAs in edible pig tissues even after 21 days post-exposure raises concerns about the safety of swine products. This was the first study to use MMPigs to elucidate the pharmacokinetics of a group of environmental pollutants. We found that MMPigs could be excellent experimental animals for toxicological studies due to their easy handling, cost efficacy for target compounds and ease of waste treatment.

VPS29-VPS35 intermediate of retromer is stable and may be involved in the retromer complex assembly process.

Retromer is a complex of proteins that functions in the endosome-to-Golgi retrieval cargo transport pathway. VPS35 works as the central subunit of retromer to recognize the cargos and binds with VPS29 and VPS26 via distinct domains. We show that deficiency of VPS35 or VPS29 accompanies degradation of other subunits, whereas VPS26 deficiency had no effect on VPS29 and VPS35 levels. Although VPS35 forms VPS26-VPS35 and VPS29-VPS35 sub-complexes with similar efficiency in vitro, VPS26-VPS35 was more easily degradable by the ubiquitin-proteasome-system than VPS29-VPS35. These results indicate that VPS29 and VPS35 form a biologically stable sub-complex in vivo.

Phosphorylcholine and SpaA, a choline-binding protein, are involved in the adherence of Erysipelothrix rhusiopathiae to porcine endothelial cells, but this adherence is not mediated by the PAF receptor.

A crucial event in the initiation of many bacterial infections is the adherence of the bacteria to host cells, and bacterial surface structures and their interactions with host cell receptors play an important role in this process. Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, which may cause acute septicemia or chronic endocarditis and polyarthritis. To study the pathogenic mechanism of the widespread vascular disease observed in the acute form of swine erysipelas, we investigated the role of phosphorylcholine (PCho), a component of the E. rhusiopathiae capsule, in bacterial adherence to porcine endothelial cells (PECs) in vitro. We found that adherence of E. rhusiopathiae strain Fujisawa to PECs was twice that of adherence to control COS-7 cells and that the adherence rates of PCho-defective mutants were approximately 30-50% lower than those of the Fujisawa strain. The adherence of the Fujisawa strain to COS-7 cells transfected with the porcine platelet-activating factor receptor (PAFR) gene, which encodes a G protein-coupled receptor that has been shown to directly bind to Streptococcus pneumoniae via PCho in the bacterial cell wall, was not enhanced. Treatment with a PAFR antagonist (WEB-2086) did not inhibit bacterial adherence to PECs. Incubation of the bacterial cells with an antibody against PCho or SpaA, a choline-binding protein anchored to PCho of the Fujisawa strain, reduced the adherence of the strain to PECs. This effect was not observed when PCho-defective mutants were used. These results suggest that E. rhusiopathiae adheres to PECs via PCho and SpaA and that the PCho-mediated adherence is independent of PAFR.

Capsule loss or death: the position of mutations among capsule genes sways the destiny of Streptococcus suis.

Streptococcus suis, an emerging zoonotic pathogen, is responsible for various diseases in swine and humans. Most S. suis strains from clinical cases possess a group of capsular polysaccharide synthesis (cps) genes and phenotypically express capsular polysaccharides (CPs). Although CPs are considered to be an important virulence factor, our previous study showed that many S. suis isolates from porcine endocarditis lost their CPs, and some of these unencapsulated isolates had large insertions or deletions in the cps gene clusters. We further investigated 25 endocarditis isolates with no obvious genetic alterations to elucidate the unencapsulation mechanisms and found that a single-nucleotide substitution and frameshift mutation in two glycosyltransferase genes (cps2E and cps2F) were the main causes of the capsule loss. Moreover, mutations in the genes involved in side-chain formation (cps2J and cps2N), polymerase (cps2I), and flippase (cps2O) appeared to be lethal; however, these lethal effects were relieved by mutations in the cps2EF region. As unencapsulation and even the death of individual cells have recently been suggested to be beneficial to the pathogenesis of infections, the results of the present study provide a further insight into understanding the biological significance of cps mutations during the course of S. suis infections.

Erysipelothrix rhusiopathiae exploits cytokeratin 18-positive epithelial cells of porcine tonsillar crypts as an invasion gateway.

Tonsils are important organs for mucosal immunity and are gateways for various pathogens, including bacteria and viruses. The purpose of the present study was to reveal how Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, invades the mucosal epithelium of the tonsils of pigs. Two germ-free piglets were orally infected with E. rhusiopathiae Koganei 65-0.15, an attenuated vaccine strain in Japan, and their tonsils of the soft palate were histologically examined four weeks after infection. Bacterial organisms were observed in dilated crypt lumens and a few epithelial cells of the crypt. Immunohistochemical examination revealed that some epithelial cells of the crypt were positive for cytokeratin (CK) 18, a specific marker for M cells in the Peyer's patches of pigs. Confocal laser scanning microscopy showed that bacterial antigens were present in the cytoplasm of CK 18-positive epithelial cells. Furthermore, an ultramicroscopic examination revealed that the bacteria-containing epithelial cells did not have microfolds or microvilli, both of which are characteristic of membranous epithelial cells (M cells), and that they were in close contact with intraepithelial phagocytes. Thus, the present observations suggest that the tonsillar crypt epithelium is a site of persistent infection for orally administered E. rhusiopathiae, and the bacteria exploit cytokeratin 18-positive epithelial cells of the crypts as portals of entry into the body.

Neurofibrillary tangles and the deposition of a beta amyloid peptide with a novel N-terminal epitope in the brains of wild Tsushima leopard cats.

Beta amyloid (Aß) deposits are seen in aged individuals in many of the mammalian species that possess the same Aß amino acid sequence as humans. Conversely, neurofibrillary tangles (NFT), the other hallmark lesion of Alzheimer's disease (AD), are extremely rare in these animals. We detected Aß deposits in the brains of Tsushima leopard cats (Prionailurus bengalensis euptilurus) that live exclusively on Tsushima Island, Japan. Aß42 was deposited in a granular pattern in the neuropil of the pyramidal cell layer, but did not form argyrophilic senile plaques. These Aß deposits were not immunolabeled with antibodies to the N-terminal of human Aß. Sequence analysis of the amyloid precursor protein revealed an amino acid substitution at the 7th residue of the Aß peptide. In a comparison with other mammalian animals that do develop argyrophilic senile plaques, we concluded that the alternative Aß amino acid sequence displayed by leopard cats is likely to be related to its distinctive deposition pattern. Interestingly, most of the animals with these Aß deposits also developed NFTs. The distributions of hyperphosphorylated tau-positive cells and the two major isoforms of aggregated tau proteins were quite similar to those seen in Alzheimer's disease. In addition, the unphosphorylated form of GSK-3ß colocalized with hyperphosphorylated tau within the affected neurons. In conclusion, this animal species develops AD-type NFTs without argyrophilic senile plaques.

Loss of capsule among Streptococcus suis isolates from porcine endocarditis and its biological significance.

Streptococcus suis, particularly serotype 2, is a pathogen of both pigs and humans associated with a wide range of diseases, including meningitis, septicaemia and endocarditis. Among the genes in the capsular polysaccharide biosynthesis (cps) locus, cps2J exists only in the serotype 2 and 1/2 strains; therefore, cps2J-positive strains are suspected to have capsules of serotype 2 or 1/2. Coagglutination using antiserotype 1 and antiserotype 2 sera and/or transmission electron microscopy analysis of 288 cps2J-positive isolates from pigs showed that 32 (100 %) isolates from meningitis were encapsulated, whereas 86 (34 %) of 256 isolates from endocarditis were unencapsulated, indicating that capsule loss often occurred in the isolates from endocarditis. To investigate the genetic backgrounds, we randomly selected 43 unencapsulated isolates and analysed their cps loci by PCR scanning. Among them, 8 and 10 isolates apparently had deletions and insertions, respectively, in cps loci. In addition, a representative unencapsulated isolate and an unencapsulated strain showed adherence to porcine and human platelets, a major virulence determinant for infective endocarditis, to a significantly greater extent than the encapsulated strains. Although the capsule is considered to be an important virulence factor in S. suis, these results suggest that loss of capsular production is beneficial to S. suis in the course of infective endocarditis.

Bovine stillbirth due to Nocardia farcinica.

A stillborn calf at 259 days of gestation was examined. The dam showed no clinical signs of disease, and the stillbirth was occurred sporadically and were characterized by focal necrosis surrounded by infiltration of the cells such as macrophages and multinucleated giant cells. The lesions were observed systemically. The hyphae were visible by Grocott's stain and they were positive by immunohistochemical stain using serum of a rabbit immunized with the isolated organism. The isolated bacteria were determined to be Nocardia farcinica by bacteriological and molecular analysis and we confirmed that the stillbirth was caused by infection with and proliferation of Nocardia farcinica. This is the first report of a bovine stillbirth caused by this organism.

Characterization of dnaJ multigene family in the cyanobacterium Synechococcus elongatus PCC 7942.

The genome of the cyanobacterium Synechococcus elongatus PCC 7942 contains four dnaJ homologs, which are classified into three types based on domain structure. Among these, dnaJ1, dnaJ2, and dnaJ3 are essential for normal growth, and hence we analyzed them with a view to characterizing their specificity. Expression analysis indicated that dnaJ2, which encodes type II DnaJ protein, exhibited typical responses to heat and high-light stresses. Their localization and ability to prevent aggregation of luciferase were also diverse, suggesting a possible functional differentiation of these proteins. Since the expression of dnaJ1, which belongs to conserved type I DnaJ, down-regulated under heat stress, the unique structure of DnaJ2 may be involved in stress responses of S. elongatus. Based on phylogenetic analysis, the diverse dnaJ family was assumed to have evolved its own specific functions in each cyanobacterial species.

Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942.

We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.