Preferential immunoglobulin oxidation in children with juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a rare chronic inflammatory disorder of the joints. There is strong evidence that oxidative damage occurs in rheumatoid diseases, including JIA. The increased level of protein oxidation products in total plasma proteins has recently been reported in children with diagnosed JIA. The objective of this study was to find out which fraction of plasma proteins is mostly damaged by oxidative stress and whether the damaging effect correlates with certain clinical or laboratory parameters. METHODS: A new approach to estimate the carbonyl content of plasma protein fractions was developed, based on two-stage electrophoresis and immunochemical detection of the carbonyl derivatives of the proteins. This method allowed us to detect and quantitate carbonyl groups in the albumin, alpha-2, beta and gamma-globulin fractions. Sera of 25 children with JIA and 13 healthy controls were tested. RESULTS: Albumin and gamma-globulins were found to be most modified by oxidation. In a group of children with systemic JIA, both albumin and gamma-globulins were oxidized while plasma gamma-globulin fraction damage was prevalent in pauciarticular JIA. CONCLUSIONS: Among plasma proteins of children with JIA, gamma-globulins were preferentially oxidized, whereas most of the other proteins did not seem to be affected. Oxidative modification of plasma proteins was correlated with the type of JIA. These findings may allow the use of carbonyls as clinical markers of inflammatory process activity in patients with different types of JIA. It is also a potential tool for monitoring oxidative protein damage in other diseases and therapies.

IbpA/B small heat-shock protein of marine bacterium Vibrio harveyi binds to proteins aggregated in a cell during heat shock.

The IbpA and IbpB are 16-kDa Escherichia coli proteins belonging to a family of small heat-shock proteins (sHsps). According to the present model, based on the in vitro experiments, sHsps are molecular chaperones that bind and prevent aggregation of nonnative proteins during heat shock. Previously, we have shown that IbpA and IbpB bind to endogenous E. coli proteins aggregated intracellularly by heat shock, which can be separated from soluble proteins and membranes in sucrose density gradients (fraction S). In this work we have found that marine bacterium Vibrio harveyi contains a single sHsp which is strongly induced by heat shock and reacts with the anti-IbpA/B serum. The 26 amino-terminal amino acids of this sHsp bear high homology to E. coli IbpA and IbpB proteins (73% and 54% identity, respectively). Fraction S was prepared from heat-shocked cells of V. harveyi, it contained high amounts of the IbpA/B protein. This result indicates that the IbpA/B protein of V. harveyi binds to the proteins that aggregate in V. harveyi cells during heat shock.

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress.

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (40H-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.

Cloning and characterization of the dnaK heat shock operon of the marine bacterium Vibrio harveyi.

We cloned the DNA region of the Vibrio harveyi chromosome containing the heat shock genes dnaK and dnaJ and sequenced them. These genes are arranged in the chromosome in the order dnaK-dnaJ, as in other proteobacteria of the alpha and gamma subdivisions. The dnaK gene is 1923 nucleotides in length and codes for a protein of 640 amino acid residues, with a predicted molecular mass of 69,076 Da and 81.2% similarity to the DnaK protein of Escherichia coli. The V. harveyi dnaJ gene has a coding sequence of 1158 nucleotides. The predicted DnaJ protein contains 385 amino acids, its calculated molecular mass is 41,619 Da and it has 74.7% similarity to the DnaJ protein of E. coli. Northern hybridization experiments with RNA from V. harveyi cells and a DNA probe carrying both the dnaK and dnaJ genes showed a single, heat-inducible transcript, indicating that these genes form an operon. Primer extension analysis revealed five heat-inducible transcriptional start sites upstream of the dnaK gene, two of which (T1 and T4) are preceded by sequences typical of the E. coli heat shock promoters recognized by the sigma 32 (sigma32) factor. Location of these promoters is highly similar to that of the E. coli dnaK promoters. No transcriptional start sites were detected upstream of the dnaJ gene. The V. harveyi dnaKJ operon cloned in a plasmid in E. coli cells was transcribed in a sigma32 dependent manner and the size of the transcript, the kinetics of transcription, and the transcriptional start sites were as in V. harveyi cells. This indicates a high conservation of the transcriptional heat shock regulatory elements between E. coli and V. harveyi, both belonging to the gamma subdivision of proteobacteria. We tested the ability of the cloned dnaKJ genes to complement E. coli dnaK and dnaJ mutants and found that V. harveyi DnaJ restored a thermoresistant phenotype to dnaJ mutants and enabled lambda phage to grow in the mutant cells. V. harveyi DnaK did not suppress the thermosensitivity of dnaK mutants but complemented the dnaK deletion mutant with respect to growth of lambda phage. V. harveyi DnaK, in contrast to DnaJ, failed to modulate the heat shock response in E. coli. Our results suggest that the DnaK chaperone may be more species specific than the DnaJ chaperone.

Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector.

groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution. The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells. In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V. harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E. coli for bacteriophage lambda growth. Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E. coli groE region. Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E. coli. This confirmed that the lambda clone indeed carries the groE region of V. harveyi. The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria. The V. harveyi cloned groE genes did not suppress thermosensitivity of the E. coli groE mutants.

Encoding-retrieval interactions in mild Alzheimer's disease: the role of access to categorical information.

Normal old adults and patients in an early phase of Alzheimer's disease (AD) were presented with photographs of common objects under two different encoding conditions: naming and naming along with category decisions. Memory was assessed with free recall, category cued recall, and recognition. For both groups, recognition was superior to cued recall which was higher than that for free recall. Most importantly, cue utilization in AD was optimized in the naming + category decision condition, although the normal old showed equivalent gains from cues following both encoding conditions. These results suggest that AD patients require more cognitive support at encoding than normal old adults to make effective use of retrieval cues. Dementia-related deficits in processing categorical information spontaneously may underlie the observed group differences in patterns of performance.

HtrA heat shock protease interacts with phospholipid membranes and undergoes conformational changes.

The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell survival only at temperatures above 42 degrees C. It has been shown by genetic experiments that HtrA is an envelope protease, functioning in the periplasmic space. To clarify the cellular localization of HtrA, E. coli cells were fractionated, and HtrA was not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protein conformation released HtrA almost completely. These results, taken together with the evidence showing that HtrA functions in the periplasm, indicate that HtrA is a peripheral membrane protein, localized on the periplasmic side of the inner membrane. As the first step toward solving the problem of HtrA-membrane interactions, the structure of HtrA in the presence of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or cardiolipin (CL) was analyzed by fluorescence and Fourier-transform infrared spectroscopy. The infrared and fluorescence data indicated an interaction of HtrA with PG and CL but not with PE suspensions. Fluorescence spectroscopy revealed that this interaction was at the level of the polar head group of the phospholipid. In the PG/HtrA system, small changes were observed in the HtrA secondary structure and a remarkable decrease of the thermal stability of the protein, which suggested changes in HtrA tertiary structure. This suggestion was supported by fluorescence data that showed a shift of the fluorescence emission spectrum of HtrA tyrosine residues in the presence of PG and a reduced fluorescence intensity, phenomena not observed in the presence of PE or CL suspensions. Infrared data revealed also that the interaction of HtrA with PG leads to a protection of unfolded protein against aggregation at relatively low temperatures. The conformational changes of HtrA in the presence of PG influenced the proteolytic activity of HtrA by increasing it at the temperatures 37-45 degrees C and inhibiting it at 50-55 degrees C. CL inhibited HtrA activity at all of the temperatures tested.

Feeling-of-knowing in fact retrieval: further evidence for preservation in early Alzheimer's disease.

The ability to retrieve and monitor factual information varying in datedness (i.e., dated vs. contemporary) was examined in healthy older adults and patients in an early phase of Alzheimer's disease (AD). Subjects were given free recall and multiple-choice recognition tests of 48 general knowledge questions. For all questions not responded to in recall, subjects made feeling-of-knowing (FOK) judgments. Results indicated dementia-related deficits in both recall and recognition, although both groups showed better recall and recognition with the dated compared with the contemporary questions. Importantly, despite deficits in fact retrieval, the AD patients showed intact monitoring of stored knowledge, as indicated by equivalent FOK accuracy for both groups. In addition, FOK accuracy was similar for the dated and the contemporary information in both groups, suggesting independence between level of general knowledge and the ability to supervise information stored in memory.

Site-directed mutagenesis of the HtrA (DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures.

The HtrA(DegP) 48-kDa serine protease of Escherichia coli is indispensable for bacterial survival at elevated temperatures. It contains the amino-acid sequence Gly208AnsSerGlyGlyAlaLeu, which is similar to the consensus sequence GlyAspSerGlyGlyProLys surrounding the active Ser residue of trypsin-like proteases. Mutational alteration of Ser210 eliminated proteolytic activity of HtrA. An identical effect was observed when His105 was mutated. The mutated HtrA were unable to suppress thermosensitivity of the htrA bacteria. These results suggest that Ser210 and His105 may be important elements of the catalytic domain and indicate that the proteolytic activity of HtrA is essential for the survival of cells at elevated temperatures.

Comparison of the structure of wild-type HtrA heat shock protease and mutant HtrA proteins. A Fourier transform infrared spectroscopic study.

The HtrA protease of Escherichia coli, identical with the DegP protease, is a 48-kDa heat shock protein, indispensable for bacterial survival only at temperatures above 42 degrees C. Proteolytic activity of HtrA is inhibited by diisopropyl fluorophosphate, suggesting that HtrA is a serine protease. We have recently found that mutational alteration of serine in position 210 of the mature HtrA or of histidine in position 105 totally eliminated proteolytic activity of HtrA. However, little was known about the consequences of the mutations on HtrA conformation. In this work, Fourier transform infrared spectroscopy has been used to examine the conformation in aqueous solution of wild-type HtrA and mutant HtrAS210 and HtrAH105 proteins. The spectra were collected at different temperatures in order to gain information also on the thermal stability of the three proteins. The analysis of HtrA protein spectrum, by resolution-enhancement methods, revealed that beta-sheet is the major structural element of the conformation of HtrA. Deconvoluted as well as second derivative spectra of wild-type HtrA and mutant HtrAS210 and HtrAH105 collected at 20 degrees C were identical, indicating no differences in the secondary structure of these proteins. The analysis of spectra obtained at different temperatures revealed a maximum of protein denaturation within 65-70 degrees C for wild-type HtrA as well as for the HtrAS210 and HtrAH105 mutant proteins. However, the thermal denaturation pattern of wild-type HtrA revealed a lower cooperativity in the denaturation process as compared to the mutant proteins which instead behaved similarly. These data suggest that the mutations in HtrA protein induced minor changes in the tertiary structure of the protein (most likely located at the mutation sites). Our results strongly support the idea that Ser210 and His105 may represent two elements of the active-site triad (Ser, His, and Asp), found in most serine proteases. We have also found that in vitro, in the range from 37 to 55 degrees C, the proteolytic activity of HtrA rapidly increased with temperature and that HtrA activity remained unchanged for at least 4 h at 45 degrees C.

Characterization of heat-shock response of the marine bacterium Vibrio harveyi.

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 degrees C was the highest temperature at which V. harveyi was able to grow steadily. A shift from 30 degrees C to 39 degrees C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15kDa protein. We showed that a protein of approximately 43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (sigma 32) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.

Effectiveness of self-generated cues in early Alzheimer's disease.

The ability to utilize cognitive support in the form of self-generated cues in mild Alzheimer's disease (AD), and the factors promoting efficient cue utilization in this group of patients, were examined in two experiments on memory for words. Results from both experiments showed that normal old adults as well as AD patients performed better with self-generated cues than with experimenter-provided cues, although the latter type of cues resulted in gains relative to free recall. The findings indicate no qualitative differences in patterns of performance between the normal old and the AD patients. For both groups of subjects, cue effectiveness was optimized when (a) there was self-generation activity at encoding, and (b) encoding and retrieval conditions were compatible.

Monitoring of general knowledge: evidence for preservation in early Alzheimer's disease.

The ability to retrieve and monitor factual information was examined in normal old adults and patients with a mild Alzheimer's disease (AD). Subjects were given free recall and multiple-choice recognition tests of 48 general knowledge questions. For both tests, subjects made confidence ratings regarding the certainty of their answers, and they also made feeling-of-knowing ratings for those questions they did not answer in recall. Results indicated a superiority of controls over patients in recall that was somewhat reduced in recognition. However, there were no group differences in any of the slopes relating rating to accuracy: recall and recognition as a function of confidence rating, and recognition of questions not answered in recall as a function of feeling-of-knowing. This pattern of outcome indicates that early AD is associated with a deficit in fact-retrieval, although the ability to monitor stored general knowledge may be intact.

arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon.

In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.

When Greta Garbo is easier to remember than Stefan Edberg: influences of prior knowledge on recognition memory in Alzheimer's disease.

This study investigated the effects of prior knowledge on recognition memory in patients with a mild Alzheimer's disease. Normal older adults and mildly demented patients were presented with dated and contemporary famous faces with name tags and were asked to generate unique statements about each person. Results indicated that both groups generated more statements about the dated than about the contemporary figures. Most important, both groups performed better with the dated than with the contemporary faces in an unexpected episodic face recognition task. This pattern of results suggests that both groups (a) possess more knowledge of dated than of contemporary famous individuals and (b) are able to utilize prior knowledge to enhance episodic remembering. Viewing these results in light of other recent work, it is concluded that differences between normal old and mildly demented individuals in the ability to utilize cognitive support for remembering may be differences in degree.

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype.

We cloned and sequenced the sohB gene of Escherichia coli. The temperature-sensitive phenotype of bacteria that carry a Tn10 insertion in the htrA (degP) gene is relieved when the sohB gene is present in the cell on a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes a periplasmic protease required for bacterial viability only at high temperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on the E. coli chromosome, precisely between the topA and btuR genes. The gene encodes a 39,000-Mr precursor protein which is processed to a 37,000-Mr mature form. Sequencing of a DNA fragment containing the gene revealed an open reading frame which could encode a protein of Mr 39,474 with a predicted signal sequence cleavage site between amino acids 22 and 23. Cleavage at this site would reduce the size of the processed protein to 37,474 Mr. The predicted protein encoded by the open reading frame has homology with the inner membrane enzyme protease IV of E. coli, which digests cleaved signal peptides. Therefore, it is possible that the sohB gene encodes a previously undiscovered periplasmic protease in E. coli that, when overexpressed, can partially compensate for the missing HtrA protein function.

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein.

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures.

We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein.