Differential cellular targets of Epstein-Barr virus (EBV) infection between acute EBV-associated hemophagocytic lymphohistiocytosis and chronic active EBV infection.

Unusual Epstein-Barr virus (EBV) infection into T or natural killer cells plays a pivotal role in the pathogenesis of acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and chronic active EBV infection (CAEBV). The precise frequency and localization of EBV genome in lymphocyte subpopulations especially within T-cell subpopulations are unclear in these EBV-related disorders. This study analyzed the frequency of EBV-infected cells in circulating lymphocyte subpopulations from 4 patients with acute EBV-HLH and 4 with CAEBV. EBV- encoded small RNA-1 in situ hybridization examination of peripheral blood lymphocytes showed a significantly higher frequency of EBV-infected cells of 1.0% to 13.4% in EBV-HLH and 1.6% to 25.6% in CAEBV, respectively. The patterns of EBV infection in lymphocyte subpopulations were quite different between acute EBV-HLH and CAEBV. EBV infection was predominant in CD8(+) T cells in all EBV-HLH patients, whereas the dominant EBV-infected cell populations were non-CD8(+) lymphocyte subpopulations in CAEBV patients. Phenotypical analysis revealed that EBV-infected cell populations from both EBV-HLH and CAEBV were activated. There was no predominance of any EBV substrain of latent membrane protein-1, EBV-associated nuclear antigen (EBNA)-1, and EBNA-2 genes between the 2 abnormal EBV-associated disorders, and self-limited acute infectious mononucleosis. These results showing differential virus-cell interactions between acute EBV-HLH and CAEBV indicated different pathogenic mechanisms against EBV infection between the 2 EBV-associated diseases, which accounts for the difference in clinical manifestations between the 2 diseases.

Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase.

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.

High precision NMR structure of YhhP, a novel Escherichia coli protein implicated in cell division.

YhhP, a small protein of 81 amino acid residues encoded by the yhhP gene in the Escherichia coli database, is implicated in cell division although the precise biological function of this protein has not been yet identified. A variety of microorganisms have similar proteins, all of which contain a common CPxP sequence motif in the N-terminal region. We have determined the three-dimensional solution structure of YhhP by NMR spectroscopy in order to obtain insight into its biological function. It folds into a two-layered alpha/beta-sandwich structure with a betaalphabetaalphabetabeta fold, comprising a mixed four-stranded beta-sheet stacked against two alpha-helices, both of which are nearly parallel to the strands of the beta-sheet. The CPxP motif plays a significant structural role in stabilizing the first helix as a part of the new type N-capping box where the Cys-Pro peptide bond adopts a cis configuration. The structure of YhhP displays a striking resemblance to the C-terminal ribosome-binding domain of translation initiation factor IF3 (IF3C). In addition, the surface charge distribution of the RNA-recognition helix of IF3C is nearly the same as that of the corresponding helix of YhhP. These results suggest a structure-based hypothesis in which binding to an RNA target plays an essential role in the function of this ubiquitous protein.

Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli.

The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.

Structural basis for the biological activity of dendrotoxin-I, a potent potassium channel blocker.

A topochemical model to explain the biological activity of dendrotoxin-I (DTX-I), a potent blocker for potassium channels, was developed by searching common spatial arrangements of functionally important residues between DTX-I, alpha-dendrotoxin, dendrotoxin-K, BgK, ShK, and charybdotoxin. The first three are structurally and functionally related to one another, and specifically target to Kv1 type potassium channels. The last three are structurally unrelated to the first three but have the ability to displace (125)I-labeled dendrotoxins on the same types of potassium channels. In order to obtain the correct electronic surface potential, thought to be crucial for the DTX-I function, we determined the three-dimensional solution structure of DTX-I by nmr spectroscopy using its correct amino acid sequence recently determined by our group. The most interesting characteristic of our model is that DTX-I has two binding sites to potassium channels: one is the cationic domain made up of Lys residues at positions 5 in the 3(10)-helix, 28 and 29 in the beta-turn, and the other is the Lys19/Tyr17/Trp37 triad located in the antiprotease domain. The cationic domain and the triad are located at the opposite sides of the molecular structure and are separated by about 25 A between Lys29 Calpha and Tyr17 Calpha. The functional triad is characterized by three distances, d(1) approximately 7.5 A (Lys19 Calpha-the center of the Tyr17 aromatic ring), d(2) approximately 8.1 A (Lys19 Calpha-the center of the 6-membered ring of the Trp37 indole group), and d(3) approximately 7. 3 A (the center of the Tyr17 aromatic ring-the center of the 6-membered ring of the Trp37 indole group). This model should aid in the pharmaceutical design of peptide and nonpeptide drugs with potassium channel blocking potencies, as well as in understanding of the physiology, pharmacology, biochemistry, and structure-function analysis of potassium channels.

Identification of the ligand-binding site of the BMP type IA receptor for BMP-4.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) >> T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.

Structure-activity relationships of calcicludine and dendrotoxin-I, homologous peptides acting on different targets, calcium and potassium channels.

Calcicludine (CaC) and dendrotoxin-I (DTX-I) possess high homology of their primary structures despite their different biological activities acting on calcium and potassium channels, respectively. In order to elucidate the channel specificity displayed by these toxins, their three-dimensional structures were compared by NMR. These analyses revealed that their overall conformations are similar except for the structure at the N-terminus. To demonstrate the significance of this N-terminal, chimeric peptides, CaC(1-30)/DTX-I(31-60) and DTX-I(1-30)/CaC(31-60), were synthesized. The CD spectra and receptor-binding measurements of chimeric peptides indicated that the contribution to the overall conformation and to the affinity of the N-terminal part of molecule seem to be more important than that of the C-terminal one. These results suggest that the N-terminal part may participate in distinguishing between calcium and potassium channels.

Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.

The guanosine binding mechanism of the Tetrahymena group I intron.

The Tetrahymena group I intron catalyzes self-splicing through two consecutive transesterification reactions, using a single guanosine-binding site (GBS). In this study, we constructed a model RNA that contains the GBS and a conserved guanosine nucleotide at the 3'-terminus of the intron (omegaG). We determined by NMR the solution structure of this model RNA, and revealed the guanosine binding mechanism of the group I intron. The G22 residue, corresponding to omegaG, participates in a base triple, G22 xx G3 x C12, hydrogen-bonding to the major groove edge of the Watson-Crick G3 x C12 pair. The G22 residue also interacts with A2, which is semi-conserved in all sequenced group I introns.

Chemical synthesis of dendrotoxin-I: revision of the reported structure.

Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled dendrotoxin binding to rat brain synaptosomal membranes.

[Dependence of axis deviation in the esophagram on computerized tomography determined wall thickness and its value in assessing depth of invasion of esophageal carcinoma]. / Abhängigkeit der Achsdeformierungen im Osophagogramm von der computertomographisch bestimmten Wanddicke und deren Wertigkeit bei der Beurteilung der Invasionstiefe des Osophaguskarzinoms.

PURPOSE: In this study we compared the abnormalities of the esophageal axis seen in the esophagogram with the thickness of the esophageal wall measured by computed tomography. We have investigated, how exactly both methods assess the local tumor invasion according to the TNM criteria and whether there is a relation between the esophageal axis and the wall thickness. METHODS: In a retrospective study we examined the esophagograms of 65 tumor patients. Computed tomography examinations were available in 40 cases. Using a graphical method the wall thickness was transferred to the esophagograms under consideration of the different scales of the images. RESULTS: There is no correlation between the different types of distortion of the esophageal axis and the wall thickness in computed tomography. However, it can be demonstrated that the distortion results from specific fixation effects with the surrounding tissue. CONCLUSIONS: Both radiological methods cannot determinate the tumor invasion correctly.

Clinicopathologic study of nasal T/NK-cell lymphoma among the Japanese.

A high prevalence of nasal lymphoma expressing a T- or natural killer (NK)-cell phenotype (NTCL) with frequent association of Epstein-Barr virus (EBV) has been indicated in Asians. To characterize NTCL among the Japanese, the clinicopathologic features of 32 cases were evaluated and the cases were also analyzed for EBV-RNA using an ISH method. Morphologically, 31 cases were identified by atypical pleomorphic lymphoid infiltrates with polymorphous, angicentric, and necrotic features. Their lymphoma cells ranged in size from small to large and were mixed in varying proportion from case to case. The other one case showed a monomorphic 'blastic' appearance. EBV-encoded small RNA (EBER) was detected in the neoplastic cells of 27 of the 32 cases examined. In the five EBV-negative cases, one was the 'blastic' type. Clonal T-cell receptor gene rearrangement was detected in none of seven cases examined. The patients had a median follow-up of 9 months (range, 1 month to 14 years and 11 months). The Kaplan-Meier estimate of overall survival was 49% at 5 years, correlating with clinical stage. These data support the concept that most cases of NTCL are identified as tumors with T/NK-cell characteristics and EBV association, distinctly different from other peripheral T-cell lymphomas. Furthermore, the one case of an EBV-negative 'blastic' variant appears not to fit well into the pleomorphic category but more closely resembles the pathologic features of extranasal angiocentric lymphoma with lymphoblastoid appearance. This study also showed no clear difference in clinical aspects other than the original site or in prognosis, between NTCL and extranasal angiocentric lymphomas despite the higher incidence of EBV association and the tendency for that peculiar anatomical site to be restricted to the former group.

[Value of the esophagogram in preoperative assessment of esophageal carcinoma]. / Stellenwert des Osophagogramms bei der präoperativen Einschätzung des Osophaguskarzinoms.

PURPOSE: In this study we utilized barium swallows exhaustively in each patient. After that we compare the results with the intraoperative findings. The goal is to select those criteria which contribute to the preoperative clinical staging of esophageal carcinoma. METHODS: In a retrospective study we examined the esophagograms of 65 tumor patients. We evaluated the location and length of the tumor, the deformation of the esophageal axis, stenosis, dilatation, and the radiological type of the tumor. All characteristics were correlated with the pathologically determined TNM-criteria, the stage of the tumor, and the palliative or curative type of resection. RESULTS: We found the following significant correlations: localisation/T-criterion, radiological type/T-and M-criteria, stenosis/type of resection, deformation of the axis, stenosis and radiological type/tumor stage. CONCLUSION: In the sense of a checklist the stenosis, the deformation of the esophageal axis and the radiological type of the tumor should be carefully evaluated and included in the report. With these data, the clinical stage of the tumor can be estimated.

[A case of progressive hemifacial and hemispheric atrophy with multiple hemi-intracerebral calcifications presenting with occipital lobe epilepsy].

A 30-year-old man with progressive hemifacial atrophy is described. He had right hemifacial atrophy and epileptic seizures first noted at the age of about 15 years. Examination revealed atrophy of the right half of the tongue, skin pigmentation in the right neck, grizzled hair on the right side of the head, and left upper temporal homonymous hemianopsia. CT and MRI revealed multiple intracerebral calcifications, and EEG showed spike discharges predominantly in the right occipital lobe, ipsilateral to the hemifacial atrophy. The epileptic seizures were associated with visual hallucinations that are characteristic of occipital epilepsy. A skin biopsy obtained from the pigmented region in the right neck showed chronic inflammatory changes consisting of severe atrophy of the epidermis, dermis, and fatty tissue, marked proliferation of collagen fibers, and perivascular infiltration by round cells and giant phagocytes. Previous descriptions on the pathogenesis of hemiatrophy of the face and brain were reviewed in relation to the present case.

The measurement of caffeine concentration in scalp hair as an indicator of liver function.

Caffeine concentration in plasma and scalp hair has been determined for subjects consuming normal daily amounts of caffeine and the results used as an indicator of individual hepatic metabolic capacity. Daily exposure to caffeine was assessed in six healthy Japanese volunteers by direct HPLC measurement of the concentrations of caffeine in aliquots of all caffeine-containing beverages consumed by the subjects. The measurements were repeated on three different occasions for each subject and caffeine consumption (mean +/- s.d.) was calculated as 178.0 +/- 84.3 mg day-1 with an intra-individual variability of 23.8 +/- 6.3% as coefficient of variation. A survey of daily caffeine consumption in 121 adult Japanese by means of a questionnaire revealed a similar value (231.8 +/- 177.8 mg day-1). Caffeine concentration in the plasma sampled during an overnight caffeine-free interval was measured by HPLC and a comparison made between healthy subjects and patients with liver disease (0.71 +/- 0.32, 0.77 +/- 0.45 and 3.92 +/- 1.91 micrograms mL-1 for healthy volunteers (n = 6), patients with hepatitis (n = 11) and those with liver cirrhosis (n = 4), respectively). Strands of scalp hair were collected from six healthy subjects and six patients with liver cirrhosis. Caffeine in hair was identified and measured by gas chromatography-mass spectrometry after digestion of the hair matrix with protease and extraction of the caffeine with chloroform. Caffeine concentration in hair collected from patients with liver cirrhosis (26.5 +/- 5.04 ng mg-1 hair) was significantly higher than that in hair sampled from healthy subjects (7.21 +/- 3.11 ng mg-1). These findings suggest that the determination of caffeine concentration in the plasma and hair of subjects consuming normal daily amounts of caffeine-containing beverages provides a practical assessment of individual liver metabolic capacity.

Clinicopathologic study of CD56 (NCAM)-positive angiocentric lymphoma occurring in sites other than the upper and lower respiratory tract.

The expression of the neural cell adhesion molecule (NCAM) (CD56, NKH-1) is a rare phenomenon in malignant lymphoma. Recently, several authors, including our group, described the clinicopathologic, phenotypic, and genotypic features of NCAM-positive tumors as a unique subgroup within a larger category of hematolymphoid malignancies. Ten cases of CD56+ angiocentric lymphoma occurring in sites other than the upper aerodigestive tract were studied for evaluating their characteristics. The disease occurred in six men and four women varying from 24 to 85 years (mean age, 53 years) who often exhibited a striking predilection for extranodal sites of involvement, such as the skin, gastrointestinal tract, and muscle, usually in the absence of peripheral lymphadenopathy. Although the cytologic appearances and immunophenotypic profile varied from case to case, these tumors often exhibited azurophilic granules, an angiocentric growth pattern, and surface CD3-, T-cell receptor (TCR) antigens-, and CD56+ phenotype without B-cell phenotype, except for a single case of CD3+, TCR alpha/beta+, and CD56+ phenotype. Genotype investigation exhibited germline configuration of the TCR beta and gamma chain genes and the immunoglobulin heavy chain gene in all five cases of surface CD3- phenotype examined, whereas the case of CD3+ phenotype showed rearrangement of TCR beta. They seem to constitute a distinct entity of the lineage spectrum spanning from natural killer (NK) cell to NK-like T cell.

Collision tumor of squamous cell carcinoma and leiomyoma in the esophagus.

Collision tumors in the esophagus are rare. In this case the collision of a leiomyoma and a squamous cell carcinoma in a 60-year-old man is presented. A possible relationship between the two tumors is discussed with reference to the literature.