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This study particularly compares the surface tensions and contact angles for molten bis(fluorosulfonyl)amide salts of imidazolium, ammonium, and phosphonium cations with the pentyl, ethoxyethyl, or ethylthioethyl group. The examined substrate plates for contact angle measurements include silicate glass, platinum, copper, graphene, and polytetrafluoroethylene (PTFE). In addition, quantum chemistry calculations were performed to obtain the optimized structures of the cations and anions of the ionic liquids (ILs) that were studied here along with some typical anions and their dipole moments, mean polarizabilities, and charge distributions. All ILs showed the same order of contact angles with respect to the substrates: PTFE > graphene ≈ copper ≈ platinum > silicate glass. By comparing the three functional groups, i.e., pentyl, ethoxyethyl, and ethylthioethyl, the ILs with the ethylthioethyl group featured a higher work of adhesion than the respective ILs with the pentyl or ethoxyethyl group. The values of the surface tensions of the ILs followed the same trend for the three functional groups. Based on the Fowkes theory, it was found that the larger surface tensions of the ILs with the ethylthioethyl group compared with pentyl and ethoxyethyl groups were because of the increase in both dispersive and nondispersive components. The quantum chemistry calculations of the ions showed a larger dipole moment and mean polarizability for the cations with the ethylthioethyl group as compared with the pentyl and ethoxyethyl groups. This is consistent with the analysis results of the surface tensions based on the Fowkes theory. By comparing other anions, the dispersive component of the surface tension of the ILs with bis(fluorosulfonyl)amide was large, which is attributed to the small dipole moment of the anion.

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLprothat possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a(YH-6) with the Rconfiguration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6suggest its potential as a lead compound for the treatment of COVID-19.

The functionalities of materials are governed by the atom type and arrangement, and perturbations caused by defects and adsorbate interactions often significantly alter the behavior of materials. Scanning probe microscopy (SPM) can capture complex interactions caused by the structures on surfaces. It is, however, difficult to analyze such interactions appearing there. In this paper, an image-processing technique that employs template matching to recognize local structures in SPM images and calculate positional correlations is reported. This approach opens new avenues for investigating intricate perturbations in the sciences and provides detailed insights into materials science.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein–protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

Amyloid fibril formation of amyloid beta peptide 1-40 (A 1-40) was reported to be retarded in the presence of 150mM phosphate buffer at pH 7 [Monji, Ustumi, Ueda, Imoto, Yoshida, Hashioka, Tashiro and Tashiro, J. Neurochemistry, 77, 1425-1432 (2007)]. In order to elucidate the reason why phosphate ion retards the amyloid fibril formation, we examined the preferential binding sites of phosphate ion to A 1-40 using chemical shift perturbation analysis of heteronuclear NMR. In titration analysis of 15N-labeled A1-40 in the presence of 150 mM phosphate ion or 150 mM chloride ion, we identified the residues affected by these ions in A 1-40. As a result, we found the tendency that phosphate ion preferentially bound to some residues located on the C-terminus region where the region was reported to be the potential -strand region in A1-40. Therefore, we suggested that phosphate ions interacted with the potential -strand region in A1-40 to be hard to form -sheet in A 1-40, resulting in retardation of the amyloid fibril formation.

Amyloid fibril formation of amyloid beta peptide 1-40 (Aβ 1-40) was reported to be retarded in the presence of 150mM phosphate buffer at pH 7 [Monji, Ustumi, Ueda, Imoto, Yoshida, Hashioka, Tashiro and Tashiro, J. Neurochemistry, 77, 1425-1432 (2007)]. In order to elucidate the reason why phosphate ion retards the amyloid fibril formation, we examined the preferential binding sites of phosphate ion to Aβ 1-40 using chemical shift perturbation analysis of heteronuclear NMR. In titration analysis of 15N-labeled Aβ1-40 in the presence of 150 mM phosphate ion or 150 mM chloride ion, we identified the residues affected by these ions in Aβ 1-40. As a result, we found the tendency that phosphate ion preferentially bound to some residues located on the C-terminus region where the region was reported to be the potential β-strand region in Aβ1-40. Therefore, we suggested that phosphate ions interacted with the potential β-strand region in Aβ1-40 to be hard to form β-sheet in Aβ 1-40, resulting in retardation of the amyloid fibril formation.

Tapes japonica lysozyme (TJL), which belongs to the invertebrate-type lysozyme family, has a unique dimer formation. The residues, which include catalytic residues (glutamate 18 and aspartate 30), at the dimer interface form electrostatic interactions. Our previous study suggested that increasing the NaCl concentration switched TJL from a dimer to monomer structure, which increased TJL activity. Therefore, conversion from the dimeric to the monomeric structure is crucial for the TJL activity. In the present study, to further understand the effect of NaCl on TJL dimer formation, we examined the protein concentration and pH dependence of TJL activity in the presence or absence of 500 mM NaCl. TJL activity was suppressed at the high protein concentration. And the optimum pH of TJL activity was decreased in the absence of NaCl. These dependencies confirm the presence of electrostatic interactions between molecules of TJL in the dimeric form in an aqueous solution.

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We have constructed a picosecond time-resolved IR dip spectrometer having a frequency resolution of < 20 cm-1and an instrument response time of 25 ps. In this system, the second harmonic of the idler wave from the OPA pumped at 800 nm and the remaining light from a regenerative amplifier (< 2.5 mJ/pulse) were differentially mixed in a KTA crystal to generate tunable high-power IR light (2750–4000 cm-1; > 60 µJ). The picosecond time-resolved IR dip spectra of phenol–(H2O)1and carbazole–(H2O)1are presented to demonstrate the capability of the constructed system. The spectral changes show clear vibrational structures of not only S0, but also S1in the 2800–3800 cm-1energy region. The system performance is also discussed.

We investigated thermal stabilities of four proteins in the presence of four kinds of sugars to analyze the mechanism of stabilization of proteins by additives. These proteins were stabilized by the addition of sugars, and the degree of stabilization correlated to the partial molar isentropic compressibility of the sugar.

In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca²⁺ binding site as in human α-lactalbumin by Escherichia coli expression system (Ser⁻¹ CaB lysozyme). In the presence of 2 mM CaCl2, the refolding yield of Ser⁻¹ CaB lysozyme at a low protein concentration (25 μg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 μg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser⁻¹ CaB lysozyme in the presence of 100 mM CaCl2 even at a protein concentration of 200 μg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser⁻¹ CaB lysozyme in the presence of various concentrations of Ca²⁺, it was suggested that the dissociation constant of Ca²⁺-peptide complex was estimated to be 20–36 mM. Moreover, the solubility of the denatured Ser⁻¹ CaB lysozyme in the presence of 100 mM CaCl2 was higher than that in the presence of 2 mM CaCl2 whereas the solubility of the denatured Ser⁻¹ lysozyme in the presence of 100 mM CaCl2 was not higher than that in the presence of 2 mM CaCl2. Therefore, it was concluded that the reduced lysozyme possessing the Ca²⁺ binding site was efficiently folded in the presence of high concentration of Ca²⁺ (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca²⁺ to the polypeptide chain in Ser⁻¹ CaB lysozyme. © 2002 Wiley Periodicals, Inc. Biopolymers 64: 106–114, 2002

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coliorigin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitroat a level similar to that seen for wild-type DnaA. When expressed at moderate levelsin vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitrohydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivodata support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.

The relation between the change in hepatitis C virus (HCV) RNA levels at the start of interferon-β (IFN-β) treatment and the long-term therapeutic response remains poorly defined. In 20 patients with chronic hepatitis C who received IFN-β (total dose 126-756 MU), the changes in serum HCV RNA during the first 2 weeks of therapy were monitored by real-time quantitative polymerase chain reaction (PCR). The serum HCV RNA level decreased rapidly during the first 24 h of therapy (first phase) and more slowly thereafter (second phase), with a mean exponential decay rate of 1.17 log10/day and 0.37 log10/day, respectively. Three patients had a sustained virologic response, 10 patients had a transient response, and 7 patients had no response. The differences in the rate of first-phase viral decline among the three groups were not significant (p = 0.21), but the differences in the rate of second-phase viral decline were significant (p = 0.0021). The mean decay rate between the end of the first 24 h and day 14 was 0.96 ± 0.43 log10/day in sustained responders, 0.39 ± 0.30 log10/day in transient responders, and 0.13 ± 0.09 log10/day in nonresponders. We conclude that during the first 2 weeks of therapy, changes in serum HCV RNA levels as monitored by real-time quantitative PCR can be used to predict the long-term response to treatment with IFN-β.

Abstract: This report describes a case of spontaneous esophageal perforation that was considered to be etiologically related to a duodenal ulcer with pyloric stenosis. The patient was a 54-year-old Japanese man who presented following the sudden onset of severe abdominal pain and dyspnea after an episode of vomiting. He had a history of duodenal ulcer. Computed tomography revealed an extremely dilated stomach containing abundant food residue, intraabdominal effusion, bilateral pleural effusion, and mediastinal emphysema, findings that strongly suggested esophageal perforation. Esophagoscopy confirmed perforation of the lower esophagus. Laparotomy revealed marked contamination, including food residue in the abdominal cavity, and a severely dilated stomach attributed to pyloric stenosis caused by a duodenal ulcer. A 2-cm longitudinal perforation was found on the right side of the lower esophagus. Because the patient's general condition was too poor to tolerate a one-stage operation (primary closure of the perforation, gastrectomy, and reconstruction), we initially performed decompression gastrostomy and control of the esophageal leakage with T-tube placement. Following the T-tube was removed 1 month later, distal gastrectomy and reconstruction of the gastrojejunostomy (Billroth II method) could be safely performed.

We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59–homoserine-105 containing Cys64–Cys80 and Cys76–Cys94. The other (Fragment II) is the peptide fragment Lys1–homoserine-58 connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115, to the peptide fragment Asn106–Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (Kd = 3.3×10–4 M at pH 8 and 25°C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64–Cys80 and Cys76–Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64–Cys80 and Cys76–Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the `safe' substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a `safe' substitution.

Abstract: A case of solitary thyroid metastasis of renal clear cell carcinoma is described. The patient was a 77-year-old Japanese woman, who was referred to our department after a thyroid tumor was identified in May 1999. She had a history of renal clear cell carcinoma of the left kidney, which had been partially resected 3 years previously. Ultrasound sonography demonstrated that a well-demarcated hypoechoic mass containing high-echo spots representing small calcifications, which measured 45 � 34 � 31 mm in size, occupied the left lobe. Computed tomography revealed a low-density mass containing small calcifications. The results of preoperative fine-needle aspiration cytology strongly suggested a clear cell carcinoma metastasizing to the thyroid. A left hemithyroidectomy was performed on July 27, 1999. A histological examination revealed that the neoplasm was composed of tumor cells with abundant clear cytoplasm and round nuclei. The histological characteristics of this thyroid tumor were virtually identical to the renal cell carcinoma resected3 years previously. Thyroglobulin stained negatively in the clear cells of the resected thyroid tumor in an immunohistochemical analysis. Clinically, the thyroid gland is a rare site of tumor metastasis; however, we should also consider the possibility of metastasis in the case of thyroid tumor patients with a history of renal cell carcinoma.

We prepared five mutant lysozymes in which glycines whose dihedral angles are located in the region of the left-handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, were mutated to an alanine residue. From analyses of their thermal stabilities using differential scanning calorimetry, most of them were more destabilized than the native lysozyme, except for the G102A mutant, which has a stability similar to that of the native lysozyme at pH 2.7. As for the destabilized mutant lysozymes, their X-ray crystallographic analyses showed that their global structures did not change but that the local structures changed slightly. By examining the dihedral angles at the mutation sites based on X-ray crystallographic results, it was found that the dihedral angles at these mutation sites tended to adopt favorable values in a Ramachandran plot and that the extent and direction of their shifts from the original value had similar tendencies. Therefore, the change in dihedral angles may be the cause of the slight local structural changes around the mutation site. On the other hand, regarding the mutation of G102A, the global structure was almost identical with that of the native structure but the local structure was drastically changed. Therefore, it was suggested that the drastic local conformational change might be effective in releasing the unfavorable interaction of the native state at the mutation site.

In order to examine how a protein can be effectively stabilized, two intramolecular cross-links, Glu35–Trp108 and Lys1–His15, which have few unfavorable interactions in the folded state, were simultaneously introduced into hen lysozyme. Both of the intramolecularly cross-linked lysozymes, 35–108 CL and 1–15 CL, containing cross-links Glu35–Trp108 and Lys1–His15, respectively, showed increases in thermal stability of 13.9 and 5.2°C, respectively, over that of wild type, at pH 2.7. On the other hand, a doubly cross-linked lysozyme showed an increase in thermal stability of 20.8°C over that of wild type, under identical conditions. Since the sum of the differences in denaturation temperature between wild type and each of the cross-linked lysozymes was nearly equal to that between wild type and the doubly cross-linked lysozyme, we suggest that the efficient stabilization of the lysozyme molecule was the direct result of the double intramolecular cross-links.

The non‐enzymatic deamidation of Asn to Asp is known to occur in proteins and peptides and is accelerated by phosphate buffer [Tyler‐Cross, R. and Schirch, V. (1991) J. Biol. Chem. 25, 22549–22556]. We attempted to identify the site in lysozyme where a phosphate ion binds by means of 1H‐15N HSQC measurements of 15N‐labeled lysozyme, which was successfully obtained using Pichia pastoris. As a result, we found that the phosphate ion was preferentially bound to Asn‐103 in hen lysozyme. The method presented here may be useful for identifying the binding site of a protein with low molecular weight substances.

Lysosomal membrane glycoproteins carry targeting information in cytoplasmic regions. Two distinct targeting motifs in these regions, GY (glycine-tyrosine) and LI (leucine-isoleucine), have been identified and characterized. Accumulating evidence suggests that the adaptor complexes (AP-1, AP-2, and AP-3) recognize this information in cytoplasmic tails of transmembrane proteins. Here we report two differentin vitroanalyses (affinity beads and surface plasmon resonance) which revealed specific interaction between the cytoplasmic tail of LGP85 and AP-1 but not so with AP-2. We also noted requirement of the LI motif of the LGP85 tail in binding to the AP-1 complex. Our data and others which indicated the binding of AP-3 to the LIMP II (synonym of LGP85) tail suggest that the cytoplasmic tail of LGP85 interacts with AP-1 at thetrans-Golgi network (TGN) and AP-3 at late endosomes, respectively. We propose that this sequential interaction between the lysosomal targeting signal and distinct APs along its transport pathway is responsible for the critical sorting of lysosomal membrane proteins and/or the potential proofreading system of mistargeted molecules.

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3have been studied by measurement of the 15N T1and T2relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rexvalues at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.

Immunotolerogenic activity of monomethoxypolyethylene glycol- (mPEG) conjugated proteins is a beneficial property in protein pharmaceutics. However, procedures for the preparation of tolerogenic mPEG proteins have not yet been defined. We prepared mPEG proteins with different mPEG contents using three proteins, hen egg lysozyme, ovalbumin and bovine gamma globulin, and their tolerogenicities to antigen-specific T and B cell responses were examined. We found the most appropriate ratio of tolerance induction to be 1.5–2.0, which is the molecular weight ratio of conjugated total mPEGs to protein. This value may assist in the preparation of tolerogenic mPEG proteins.

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We prepared three peptide fragments (fg.59-105, fg.63-105 and fg.64-105) by the BrCN cleavage of mutant lysozymes where Ile58, Trp62 and Trp63 were mutated to Met, respectively. From the analysis of formation of the disulfide bonds among Cys64, Cys76, Cys80 and Cys94 in the renaturation of each peptide fragment from the reduced form, Trp62 and Trp63 were required for the effective formation of two disulfide bonds. Especially, Trp62 was found to be involved in the correct formation of the disulfide bonds.

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

Abstract:: We describe a patient, 54-year-old woman, with lymphangiomas of the jejunum and mesentery presenting as acute abdomen. She had sudden onset of severe abdominal pain with guarding and fever. Physical examination revealed an elastic hard mass, with marked tenderness in the right abdominal region. Ultrasonography and computed tomography revealed a heterogenous mass, 20 � 15 � 8 cm in size, in the mesentery. An emergency operation was performed with the diagnosis of diffuse peritonitis due to an inflammatory tumor in the mesentery. At operation, the tumor was found in the mesentery, adhering to a 60-cm segment of the jejunum. Multiple small tumors were seen in the submucosal layer of jejunum. The tumor was excised with a 60 cm length of the jejunum. Histologically, the tumors were diagnosed as cavernous lymphangiomas. Although lymphangioma in the abdomen is rare in adults, we should consider it as one of the causes of acute abdomen.

Lys96 and Lys97 in lysozyme are located at the C terminus of α-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8·0 and 37°C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from α-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Tro63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of α-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme. Copyright 1994, 1999 Academic Press

Myocardial mitochondrial function after acute adriamycin exposure was compared in infant and adult mice. Heart mitochondria were isolated 48 h after an intraperitoneal injection of adriamycin. Concentrations of adriamycin in serum and heart tissue were not significantly different between infant and adult mice. Oxygen consumption (state 3 respiration), and respiratory control ratio (RCR) were studied polarographically. Enzyme activities in the respiratory chain [succinate-cytochrome c reductase (SCCR), NADH-cytochrome c reductase (NCCR), cytochrome c oxidase (CCO)], and adenine nucleotide translocase (ANT) were assayed. After saline injection (control), no significant differences were detected in state 3 respiration, RCR, and enzyme activity of ANT between infant and adult mice. The respective enzyme activities of SCCR, NCCR, and CCO in adult mice were significantly lower than those in infant mice. After adriamycin injection in adult mice, there were significant decreases in state 3 respiration (using glutamate and malate as substrates from 239 ± 25 to 160 ± 50 nanoatom O2/min/mg protein), RCR (using glutamate and malate as substrates from 7.2 ± 1.0 to 4.4 ± 1.4), and enzyme activities of SCCR (from 279 ± 30 to 178 ± 28 nmol/min/mg protein) and NCCR (from 331 ± 43 to 237 ± 30 nmol/min/mg protein), but there were no significant changes in infant mice. No significant changes in enzyme activities of CCO and ANT were found in either infant or adult mice following the administration of adriamycin. In conclusion, adriamycin is less toxic on the myocardial mitochondrial function in infant mice than in adult mice.

Abstract: We describe a case of mucinous cystadenoma of the appendix with perityphlic granuloma. The patient developed a hard palpable mass in the right lower quadrant and barium enema inducted irregular bowel wall at the cecum. Based on a preoperative diagnosis of carcinoma of the appendix, we performed a laparotomy. On operation, we found a tumor mass in the region of the appendix; the mass adhered strongly to the retroperitoneum and cecum. Right hemicolectomy was carried out on a diagnosis of carcinoma of the appendix. Microscopic examination revealed mucinous cystadenoma of the appendix with perityphlic granuloma. Mucinous cystadenoma in the appendix is a rare tumor. In this patient, the tumor was accompanied by granuloma formation in adjacent tissues because of mucin expelled from the appendix. This case emphasizes that granuloma formation can make of difficult to differentiate mucinous cystadenoma in the appendix from cancer.

We examined the usefulness of factor analysis for the diagnosis of hepatocellular carcinoma by analysis of the data obtained by radionuclide angiography. The data could be separated into two factors, a hepatic phase (hepatic artery and portal vein) and an arterial phase (aorta and kidney). In patients with hepatocellular carcinoma, the factor of the tumor is included in the arterial phase, so that the cancerous region could be differentiated from the noncancerous region. The ratio of the radioactivity of the cancerous region to the noncancerous region was used to estimate the blood flow of the tumor region.

Site-directed mutagenesis was used to identify amino acid residues essential for the thermostability of the DNA-binding protein HU from the thermophile Bacillus stearothermophilus(BstHU). Two mutants,BstHU-A27S and BstHU-V42I, in which Ala27and Val42in BstHU were replaced by the corresponding amino acids Ser27and Ile42, respectively, in the homologue from a mesophileB. subtilis(BsuHU), were less stable than the wild-type BstHU (63.9 °C), showing Tmvalues of 58.4 °C and 60.1 °C, respectively, as estimated by circular dichroism (CD) analysis at pH 7.0. The denaturation of two mutants was further characterized using differential scanning calorimetry; the Tmvalues obtained by calorimetric analysis were in good agreement with those estimated by CD analysis. The results suggest that Ala27and Val42are partly responsible for enhancing the thermostability of BstHU. When considered together with previous results, it is revealed that Gly15, Ala27, Glu34, Lys38, and Val42are essential for the thermostability of thermophilic protein BstHU. Moreover, five thermostabilizing mutations were simultaneously introduced into BsuHU, which resulted in a quintuple mutant with a Tmvalue of 71.3 °C, which is higher than that of BstHU, and also resulted in insusceptibility to proteinase digestion.

An alkaline fraction separated by ion exchange chromatographies from the water extract of PANAX GINSENG root stimulated the proliferation of baby hamster kidney-21 cells. Separation of the alkaline fraction by MCI-gel CHP 20P column chromatography followed by dialysis provided an active material. By a reversed-phase HPLC the active material was separated into six fractions, and an active colorless Compound 1 was obtained from fraction 2 in a pure State. Compound 1 was composed of the following amino acids; Gly, Arg, Glu, Val in a ratio of 1:1:1:1, and caused 20% enhancement of proliferation of BHK-21 cells at a concentration of 3.40 M. On the basis of physical and spectral data the structure of Compound 1 was established as a tetrapeptide, Gly-Arg-?-Glu-Val-NH 2.

Abstract: TheLimulus amebocyte lysate (LAL) test has the disadvantage of being influenced by various inhibitors and activators. We have developed a method for the LAL reaction that involves the specific adsorption and isolation of endotoxin using a membrane filter unit and immobilized histidine; in this present study we used the method to measure endotoxin in the plasma of patients with acute or chronic liver disease. The adsorbed endotoxins are separated from LAL-inhibitors or-activators by the membrane filter unit, and their activity is directly assayed with the LAL reagent in a filter cup without any inhibition or activation. The study population consisted of 23 subjects, 3 with fulminant hepatitis and 20 with cirrhosis (9 with esophageal varices and 11 without). All 3 (100%) of the samples of plasma from patients with fulminant hepatitis were positive for endotoxin, as were the samples of 7 (78%) of the 9 patients with cirrhosis and esophageal varices, and 2 (18%) of the 11 patients with cirrhosis but without such varices. The results suggested that this method appears to be useful for assaying the concentration of endotoxin in patients with fulminant hepatitis or cirrhosis of the liver.

We report a method by which the contribution of the superior mesenteric vein to the portal blood flow can be evaluated noninvasively. An enteric-coated capsule containing [123I]iodoamphetamine is given by mouth 3 h before the examination. The data obtained are treated by computer to calculate the portal shunt index (SI) through the superior mesenteric vein. The SI was higher for more severe liver disorders, Increasing in the order of chronic persistent hepatitis, chronic aggressive hepatitis, and cirrhosis. The SI was higher in cirrhotic patients than in chronic hepatitis patients or healthy volunteers (both,P<0.001). The SI was higher in cirrhotic patients with esophageal varices than in such patients without varices (P<0.05). The SI was higher in cirrhotic patients with ascites than in such patients without ascites (P<0.001). The SI was higher in cirrhotic patients with encephalopathy than in those without encephalopathy (P < 0.01). Correlation was significant between the SI and classical indicators of functional reserve. This method is clinically useful.

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Approaches to improving the functionality of lysozyme are presented. Lysozyme was variously modified and the stabilities of the derivatives were determined by thermal denaturation experiments. Contributions of salt bridge(s), hydrophobic interactions(s), and cross-linkage(s) were evaluated. The stabilities against proteolysis were also considered. For the latter stability, it might be important to depress the rate of unfolding, i.e., to stabilize the native conformation. As a rule, salt bridges and hydrophobic interactions stabilize the native conformation and cross-linkages destabilize the denatured conformation. However, cross-linkages are apt to introduce strains in the native conformation and only suitable lengths of cross-linkages can stabilize the protein. The stabilization was shown to be generally effective in improving the functionality of proteins. Catalytic groups in lysozyme (Glu-35 and Asp-52) were variously modified and finally converted to the respective amides. The participation of these groups in the catalytic function was confirmed. The specificity of lysozyme was modified. Asp-101, which lies on the top of the active site cleft of lysozyme, was variously modified and the effects on the hydrolysis patterns of a hexamer of N-acetylglucosamine were analyzed. Some approaches to endowing lysozyme with altered functions are also presented. In order to give higher esterase activity to lysozyme, the complementarity of enzyme and substrate was investigated by modifying substrate and the active site cleft of lysozyme. An attempt was made to convert lysozyme into a transaminase by introducing pyridoxamine to the active site cleft of lysozyme. Finally, we have started to apply genetic engineering to this kind of investigation and would like to see how far we can go with protein engineering to improve the nature of proteins.

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.

Objective Regulator of calcineurin 1 (RCAN1) controls plasticity of the nervous system and depressive conditions by regulating brain-derived neurotropic factor (BDNF) and plays a crucial role in neural and cardiac pathways. The apolipoprotein E gene (ApoE) is a robust risk factor for progression of Alzheimer’s disease. A fatty diet is considered detrimental for metabolic disorders, such as obesity and cardiovascular diseases.Methods We examined the neuronal and cardiac protective roles of RCAN1 in ApoE−/− mice that were fed a high- or low-fat diet with and without voluntary movement for 3 months. Organ weights, laboratory data, histology, RNA expression, and behavior were examined.Results A high-fat diet with exercise improved depressive function, as examined by the forced swimming test, and RCAN1mRNA expression was induced in the hippocampus. A low-fat diet with exercise resulted in a reduced body weight, higher heart weight/body weight ratio, and lower circulating triglyceride levels compared with a low-fat diet without exercise. RCAN1mRNA expression was increased in cardiomyocytes in ApoE−/− mice.Conclusions The combination of a high-fat diet and exercise might reduce depressive function, whereas a low-fat diet with exercise leads to cardioprotection. Induction of RCAN1expression might affect neuroplasticity and cardiac function.