A carboxyl-terminal Cys2/His2-type zinc-finger motif in DNA primase influences DNA content in Synechococcus PCC 7942

The DNA primase gene, dnaG, has been isolated from the cyanobacterium Synechococcus PCC 7942. It is not part of a macromolecular synthesis operon but is co-transcribed with pheT and located adjacent to the metallothionein divergon, smt. At the carboxyl terminus of this DnaG is a Cys2/His2 zinc-finger motif. The carboxyl-terminal 91 residues bound 65Zn and 0.95 g atom of Zn2+ mol-1 were detected with 4-(2-pyridylazo)resorcinol. Following exposure to Cd2+, 0.95 g atom of Cd2+ was displaced by 2 equivalents of p-(hydroxymercuri) phenylsulfonate mol-1, while only 0.03 g atom of Cd2+ was displaced mol-1 polypeptide missing the carboxyl-terminal (residue 592 onward) zinc-finger motif. Zn2+ caused an increase in intensity, and a reduction in wavelength, of Trp fluorescence at the tip of the predicted zinc-finger, while EDTA caused the converse. Cells containing a single chromosomal codon substitution (C597S), altering the zinc-finger, were generated by exploiting Zn2+-sensitive smt mutants and the proximity of dnaG to smt. Cells in which smt and dnaG(C597S) had integrated into the chromosome were selected via restored Zn2+ tolerance. Synechococcus PCC 7942 and its dnaG(C597S) mutant grew at equivalent rates, but the latter had a reduced number of chromosomes.     

Construction of a family of Cys2His2 zinc binding sites in the hydrophobic core of thioredoxin by structure-based design

A semi-automated, rational design strategy has been used to introduce a family of seven single, mononuclear Cys2His2 zinc sites at various locations in the hydrophobic core of Escherichia colithioredoxin, a protein that is normally devoid of metal centers. The electronic absorption spectra of the CoII complexes show that five of these designed proteins bind metal with the intended tetrahedral geometry. The designed sites differ in their metal-binding constants and effects on protein stability. Since these designs are constructed within the same host protein framework, comparison of their behavior allows a qualitative evaluation of dominant factors that contribute to metal-binding and metal-mediated protein stabilization. Metal-binding constants are dominated by steric interactions between the buried, designed coordination sphere and the surrounding protein matrix. Metal-mediated stability is the consequence of differential binding to the native and unfolded states. Increased interactions with the unfolded state decrease the stabilizing effect of metal binding. The affinity for the unfolded state is dependent on the placement of the primary coordination sphere residues within the linear protein sequence. These results indicate that a protein fold can have a remarkably broad potential for accommodating metal-mediated cross-links and suggest strategies for engineering protein stability by constructing metal sites that maximize metal binding to the native state and minimize binding to the unfolded state.     

Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein

Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.     

Heat shock protein 70 family: multiple sequence comparisons, function, and evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus "matches" 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins.     

Long-term survival of 2 cases of hemochromatosis respectively homozygous for His63Asp and Cys282Tyr mutations]

A 45-year-old Greek patient was found to have a moderate iron overload (ferritin 1213 micrograms/l, serum iron 21.5 mumol/l, transferrin saturation 40%). He underwent 12 phlebotomies of 450 cc over an 8-year period and ferritin was normalized (267 micrograms/l) after the seventh. Study of the HLA-H gene in leukocyte DNA showed that the patient is homozygous for the His63Asp mutation while no modification was found at position 282. This case is compared with that of a 50-year-old Swiss male presenting a severe iron overload (ferritin 7660 micrograms/l, serum iron 36.5 mumol/l, transferrin saturation 97%). Although this patient has undergone 77 phlebotomies (450 cc each time) over a 2-year period, he continues to have a high ferritin level (2200 micrograms/l). HLA-H gene analysis showed the absence of codon 63 mutation and the presence of Cys282Tyr mutation in the homozygous state. The study of these two cases indicates that penetrance of the His63Asp mutation in the homozygous state is very low as compared to Cys282Tyr and results in moderate iron accumulation, probably without organ damage. This genotype must be looked for whenever moderate iron overload is present.     

A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein

Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.     

The Mof2/Sui1 protein is a general monitor of translational accuracy

Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed -1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene. mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed -1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.     

Communication in general practice: recognition and treatment of mental illness

The scope of the present review is to describe epidemiology, classification, symptomatology and treatment of diabetic peripheral somatic neuropathy and autonomic neuropathy. Special attention is paid to the use of local anaesthetic agents in painful diabetic neuropathy. Denervation hypersensitivity is a characteristic of autonomic neuropathy in diabetic patients. The pathophysiology behind this phenomenon is elucidated in this review and most recent studies related to diabetic encephalopathy are reviewed. References for this review were acquired via a MedLine and MedLars literature search.     

Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.     

Evolution of the arthropod prophenoloxidase/hexamerin protein family

PURPOSE: Because intraperitoneal (i.p.) therapy may provide a therapeutic advantage and because hyperthermia enhances carboplatin (CBDCA) cytotoxicity, we evaluated the feasibility, toxicity, and pharmacokinetics of CBDCA given via continuous hyperthermic peritoneal perfusion (CHPP) in patients with small-volume residual ovarian cancer. Patients and METHODS: Six patients underwent optimal cytoreductive procedures (residual disease < or =5 mm) as initial treatment of stages II and III epithelial ovarian adenocarcinoma. All patients received a 90-min CHPP at a CBDCA dose of 800-1200 mg/m2, with the perfusate being recirculated rapidly from a reservoir through a heat exchanger, resulting in i.p. temperatures of 41-43 degrees C. Plasma, perfusate, and urine samples were collected and platinum was quantified by flameless atomic absorption spectrophotometry. RESULTS: At no time did any patient's core temperature exceed 40 degrees C. Peak perfusate platinum concentrations were 8- to 15-fold higher than peak ultrafilterable plasma concentrations. The permeability-area product was extremely high and variable (14-90 ml/min), resulting in a regional advantage of 1.9-5.3. The percentage of the dose absorbed ranged widely from 27% to 77%. Dose-limiting hematologic toxicity was observed at a dose of 1200 mg/m2 and this was associated with a CBDCA AUC in plasma of 11 mg min ml(-1). CONCLUSION: CHPP with CBDCA was safely given to three patients at a dose of 800 mg/m2, and dose-limiting hematologic toxicities observed at 1200 mg/m2, correlated with the plasma CBDCA exposure established when lower doses of CBDCA are given systemically. The pharmacokinetic data are consistent with the expected effect of vigorous mixing on the exposed peritoneal surface area. Variable drug absorption and clearance make the prediction of systemic exposure highly uncertain. These findings may have important implications for novel therapies given i.p.     

Evolution of the alpha-crystallin/small heat-shock protein family

The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.     

Dopamine receptor D2 Ser/Cys311 variant associated with disorganized symptomatology of schizophrenia

AIMS: To investigate the prevalence of lymphocytic gastritis in patients with coeliac disease. METHODS: Gastric biopsies from 70 patients with coeliac disease were examined by light microscopy for the presence of lymphocytic gastritis, defined as 25 or more intraepithelial lymphocytes/100 gastric columnar epithelial cells. RESULTS: Lymphocytic gastritis was found in seven cases. Positive cases had a mean of 32.1 intraepithelial lymphocytes/100 columnar cells, compared with a mean of 13.9 in negative cases, and 5.15 in noncoeliac controls. No differences were found for age, sex, gastric corpus or antrum, or degree of inflammation in the gastric lamina propria. All intraepithelial lymphocytes were of T cell lineage. Cases not showing lymphocytic gastritis did however show significantly increased gastric intraepithelial lymphocytes compared with non-coeliac controls. Eighteen of 70 cases were positive for Helicobacter pylori, and four of seven cases of lymphocytic gastritis were H pylori positive; no significant difference was observed between H pylori positive and negative patients. Three cases had concomitant ulcerative enteritis, of which none showed lymphocytic gastritis, while five cases had concomitant enteropathy associated T cell lymphoma, of which one showed lymphocytic gastritis. CONCLUSIONS: Lymphocytic gastritis occurred in 10% of patients with coeliac disease. Cases without lymphocytic gastritis nevertheless showed increased gastric intraepithelial lymphocytes. Coeliac disease may on occasion be a diffuse lymphocytic enteropathy occurring in response to gluten. Lymphocytic gastritis outside coeliac disease may involve an immune response to luminal antigens, such as H pylori, not unlike the response to gluten in patients with coeliac disease.     

The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait

Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.     

Molecular evolution of the family Camelidae: a mitochondrial DNA study

We report the first molecular evolutionary analysis of the family Camelidae by analysing the full DNA sequence of the mitochondrial cytochrome b gene. Estimates for the time of divergence of the Old World (Camelini) and New World (Lamini) tribes obtained from sequence data are in agreement with those derived from the fossil record. The DNA sequence data were also used to test current hypotheses concerning the ancestors of the domesticated llama and alpaca. The results show that hybridization has occurred in the ancestry of both domesticated camelids, obscuring the origin of the domestic species.     

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.