Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.

Complete genome sequence of Treponema pallidum, the syphilis spirochete.

The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

Changes in the stability of a human H3 histone mRNA during the HeLa cell cycle.

A major component of the regulation of histone protein synthesis during the cell cycle is the modulation of the half-life of histone mRNA. We have uncoupled transcriptional and posttranscriptional regulation by using a Drosophila hsp70-human H3 histone fusion gene that produces a marked human H3 histone mRNA upon heat induction. Transcription of this gene can be switched on and off by raising and lowering cell culture temperatures, respectively. HeLa cell lines containing stably integrated copies of the fusion gene were synchronized by double thymidine block. Distinct populations of H3 histone mRNA were produced by heat induction in early S-phase, late S-phase, or G2-phase cells, and the stability of the induced H3 histone mRNA was measured. The H3 histone mRNA induced during early S phase decayed with a half-life of 110 min, whereas the same transcript induced during late S phase had a half-life of 10 to 15 min. The H3 histone mRNA induced in non-S-phase cells is more stable than that induced in late S phase, with a half-life of 40 min. Thus, the stability of histone mRNA is actively regulated throughout the cell cycle. Our results are consistent with an autoregulatory model in which the stability of histone mRNA is determined by the level of free histone protein in the cytoplasm.

Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product.

We have previously shown that the human 70-kilodalton heat shock protein gene (hsp70) is induced by the adenovirus E1A gene product and during the S-G2 phase of the cell cycle. In this study, we investigated the effect of E1A on the expression of other human hsp genes. A gene encoding one form of the hsp89 protein (hsp89 alpha) was activated during an adenovirus infection with kinetics similar to those of activation of hsp70. The induction required a functional E1A gene. However, the hsp89 transcript was not cell cycle regulated. Genes encoding another form of hsp89 and the hsp27 protein were not induced by E1A or during the cell cycle. Further examination of hsp70 expression revealed a greater complexity than previously seen. S1 nuclease analysis using an hsp70 cDNA as well as a distinct hsp70 genomic clone demonstrated three related hsp70 transcripts; two were induced by E1A, and one was not. Both of the E1A-inducible genes were regulated during the cell cycle. All three were induced by heat shock. These results suggest common aspects of control among certain members of this family of cellular genes distinct from heat shock control. Finally, using viruses that express the individual E1A proteins, we found that the hsp70 gene is induced by the 12S and the 13S E1A products. The efficiency of induction by the 12S product was somewhat less than that by the 13S product but only by a factor of less than 2. This is in contrast to the induction of early viral genes, for which the 13S product is considerably more efficient than the 12S product.

The Comprehensive Microbial Resource.

One challenge presented by large-scale genome sequencing efforts is effective display of uniform information to the scientific community. The Comprehensive Microbial Resource (CMR) contains robust annotation of all complete microbial genomes and allows for a wide variety of data retrievals. The bacterial information has been placed on the Web at http://www.tigr.org/CMR for retrieval using standard web browsing technology. Retrievals can be based on protein properties such as molecular weight or hydrophobicity, GC-content, functional role assignments and taxonomy. The CMR also has special web-based tools to allow data mining using pre-run homology searches, whole genome dot-plots, batch downloading and traversal across genomes using a variety of datatypes.

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Stress response protein (srp-27) determination in primary human breast carcinomas: clinical, histologic, and prognostic correlations.

Expression of an estrogen-regulated protein known as the 27,000-d heat-shock or stress-response protein (srp-27) was evaluated in human breast carcinomas and established breast cancer cell lines. Results obtained by Northern and Western blot analyses and immunohistochemical methods were concordant. Immunohistochemical assessment of srp-27 expression in 300 breast carcinomas (with median patient follow-up of 8 years) was performed. Twenty-six percent of lymph node-negative and 45% of lymph node-positive tumors were overexpressors. Univariate analysis demonstrated significant correlations between srp-27 overexpression and estrogen receptor (ER) content, pS2 protein expression, nodal metastases, advanced T stage, lymphatic/vascular invasion, and a shorter disease-free survival period (but not a shorter overall survival) for the study population as a whole. Regression tree analysis showed that srp-27 expression was an independent prognostic indicator for disease-free survival only in patients with one to three positive lymph nodes. The Cox proportional hazards model confirmed the independent prognostic significance of nodal involvement, T stage, and ER content but failed to recognize srp-27 overexpression as a significant independent parameter predictive of patient outcome in the patient population as a whole. The observed associations between srp-27 overexpression and more aggressive tumors suggest a biologic role for srp-27 in human breast carcinomas.

Effect of overexpression of the small heat shock protein HSP27 on the heat and drug sensitivities of human testis tumor cells.

In contrast to most metastatic cancers, testicular germ cell tumors are cured in more than 80% of patients using cisplatin-based combination chemotherapy. Testis tumor cells in vitro retain their sensitivity to chemotherapeutic drugs, radiation, and other stresses, such as heat shock. Having shown that this is associated with low constitutive levels of heat shock protein (HSP) 27, we determined the effect of overexpression of HSP27 on the heat and drug sensitivities of a human testis tumor cell line, 833K. Cells were cotransfected with plasmids containing a neomycin resistance gene and the full-length human HSP27 gene, and four clones that overexpressed HSP27 by factors of 3.7-38.3-fold compared with the parental cells were selected. The overexpressing cells were more resistant to heat shock, cisplatin, and doxorubicin, and this was associated with modest increases (17-30%) in population doubling times and a small reduction in the number of S-phase cells. These results suggest that the low constitutive levels of HSP27 in testis tumor cells may contribute to the sensitivity of testicular germ cell tumors to chemotherapy, and that targeting HSP27 may improve response rates in other types of cancer.

Cloning and sequencing of the Legionella pneumophila fur gene.

Iron is required for the intracellular and extracellular growth of Legionella pneumophila (Lp). In addition, variations in iron levels may serve as a signal for changes in gene expression. In a number of bacterial pathogens, the regulation of gene expression by iron is usually mediated by the Fur (ferric uptake regulation) repressor protein. Through complementation of an Escherichia coli fur mutation and nucleotide sequence analysis, we have cloned and characterized the Lp fur gene. Lp fur encoded a 15.0-kDa protein whose repressive activity was, as expected, highest in bacteria grown in iron-rich media. Computer analysis determined that Lp Fur had an amino-acid identity of over 54% and a similarity of over 72% to the Fur of E. coli, Yersinia pestis, Vibrio species and Pseudomonas aeruginosa. The promoter region of Lp fur contained sequences homologous to the Fur-binding site, suggesting that fur is autoregulated in Lp. Finally, Southern blot hybridizations demonstrated that fur is conserved among Lp strains and Legionella species.

Cloning and sequencing of a cDNA encoding the canine HSP27 protein.

The nucleotide (nt) sequence encoding a 27-kDa heat-shock protein (HSP27) was determined from cDNAs cloned from a canine smooth muscle library. The primary structure deduced from the nt sequence reveals a 209-amino-acid protein having 86-89% identity with human, mouse, rat and hamster small HSP. Similar to human HSP27, the canine protein contains three Ser residues that are potential MAPKAP kinase II substrates.

Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia.

Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89-kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA.

Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death.

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Influence of phosphorylation and oligomerization on the protective role of the small heat shock protein 27 in rat adult cardiomyocytes.

Recent reports have demonstrated that the heat shock proteins (hsp) and in particular the hsp70 confer protection against cardiac ischemic damage. More recently, we have shown that increased expression of another heat shock protein, the hsp27, through an adenovirus vector system protects adult cardiomyocytes against ischemic injury. This small heat shock protein undergoes phosphorylation when the cell is under stress. This has led many to speculate that phosphorylation of hsp27 is required for the protective role this protein plays in the cell. In order to investigate this possibility, we have mutated the serines that are the sites of phosphorylation on the hsp27, to glycines or alanines. These nonphosphorylatable mutants of hsp27 were cloned into adenoviral vectors and used to infect adult rat cardiomyocytes to assess their ability in protecting against ischemic injury. In addition, we used a specific inhibitor of p38 MAP kinase that is a key member of the kinase pathway responsible for phosphorylating the hsp27. Our present results show that the nonphosphorylated hsp27 forms larger oligomeric complexes than the phosphorylated hsp27. Interestingly, phosphorylation of hsp27 seems not to play a role in its ability to protect adult rat cardiomyocytes against ischemic damage.