Successful tocilizumab and tacrolimus treatment in a patient with rheumatoid arthritis complicated by systemic lupus erythematosus.

We report a 37-year-old female of intractable rheumatoid arthritis (RA) complicated by systemic lupus erythematosus (SLE), who was successfully treated with a combination of tocilizumab (TCZ) and tacrolimus. She was diagnosed with RA when she was 21 years old, and was administered oral prednisolone, injectable gold and salazosulfapyridine, but deformity of her hands gradually developed. She developed high fever and thrombocytopenia when she was 35 years old. Renal involvement, pericarditis, positive antinuclear antibody and high level of anti-double-stranded DNA antibody were found and the patient was diagnosed with SLE. Polyarthritis and immunological abnormalities developed despite aggressive immunosuppressive therapy including high-dose corticosteroids and intravenously administered cyclophosphamide. Tacrolimus (TAC) therapy gave only partial improvement of joint symptoms. After the initiation of combination therapy with TCZ, not only was a complete remission of RA obtained, but also the serum levels of SLE markers dramatically decreased. Our report suggests the possibility that this combination therapy is effective in treating SLE as well as RA.

A cross-section of the fitness landscape of dihydrofolate reductase.

In vitro molecular evolution is regarded as a hill-climbing on a fitness landscape in sequence space, where the 'fitness' is a quantitative measure of a certain physicochemical property of a biopolymer. We analyzed a 'cross-section' of the enzymatic activity landscape of dihydrofolate reductase (DHFR) by using a method of analysis of a fitness landscape. We limited the sequence space of interest to the five-dimensional sequence space, where the coordinate corresponds to the 1st, 16th, 20th, 42nd and 92nd site in the DHFR sequence. Thirty six mutants mapped into the limited sequence space were taken in the analysis. As a result, the cross-section is of the rough Mt Fuji type based on the mutational additivity. The ratio of the mean slope to the roughness is 2.8 and the Z-score of the original ratio against a distribution of random references is 7.0, which indicates a large statistical significance. The existence of such a cross-section was discussed in terms of the occurrence probability of sets of five sites distantly separated from each other on the DHFR 3D structure. Our results support the effectiveness of the evolution strategy which exploits the accumulation of advantageous single point mutations in such a cross-section.

An approach for protein to be completely reversible to thermal denaturation even at autoclave temperatures.

Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on-off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120 degrees C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.

ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.

Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

Systematic circular permutation of an entire protein reveals essential folding elements.

The importance of chain connectivity in determining protein function and stability can be examined by breaking the peptide backbone using a technique such as circular permutation. Cleavage at certain positions results in a complete loss of the ability of the protein to fold. When such cleavage sites occur sequentially in the primary structure, we call the region a 'folding element', a new concept that could assist in our understanding of the protein folding problem. The folding elements of dihydrofolate reductase have been assigned by conducting a systematic circular permutation analysis in which the peptide backbone was sequentially broken between every pair of residues in the protein. The positions of folding elements do not appear to correspond to secondary structure motifs, substrate or coenzyme binding sites, or accessible surface area. However, almost all of the amino acid residues known to be involved in early folding events are located within the folding elements.

Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein.

The urea-induced equilibrium unfolding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left harpoon I1 right harpoon over left harpoon I2 right harpoon over left harpoon U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P. J., Bilsel, O., and Matthews, C. R. (1999) Protein Sci. 8, 1623-1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. Size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of approximately 24 A for the native state of alphaTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 A, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at approximately 3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from approximately 38 to approximately 52 A. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self-association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to approximately 54 A at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical for the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N right harpoon over left harpoon I1 and I1 right harpoon over left harpoon I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 right harpoon over left harpoon U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincón, G., Gualfetti, P. J., and Matthews, C. R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respective conformational ensembles.

[Surgical technique and long-term follow-up of laminoplasty using titanium miniplates].

Problems concerning a spinal deformity due to simple laminectomy and the usefulness of laminoplasty are often reported recently. In our hospital, laminoplasty with titanium miniplates for spinal operations has been used in all cases since 1992. We started to use this method after we encountered several cases of post-operative spinal deformity following multiple-level laminectomy. We reviewed two different subgroups of patients. Group A consisted of 38 cases who were operated on using simple laminectomy. Group B consisted of 32 cases who were operated on using laminoplasty with titanium miniplates. Postoperative spinal deformity occurred in 5 patients of Group A (13%), and in none of Group B (0%). In particular, postoperative spinal deformity occurred in one child case of Group A (33%), and in none of Group B (0%). Laminoplasty is a very simple and efficient technique, and advantages are the presentation of stability of the posterior column and curvature of the laminae, as well as protection of the spinal cord. Compatibility with magnetic resonance imaging (MRI) is another advantage of titanium miniplates, because long follow-up using MRI is always necessary in these cases. In conclusion, laminoplasty with titanium miniplates for spinal operations are a very useful and safe technique.

Circular permutation analysis as a method for distinction of functional elements in the M20 loop of Escherichia coli dihydrofolate reductase.

A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia coli dihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1-4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate in E. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of alpha carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following alpha helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates.

Effect of clot removal and surgical manipulation on regional cerebral blood flow and delayed vasospasm in early aneurysm surgery for subarachnoid hemorrhage.

BACKGROUND: Effect of clot removal and surgical manipulation on cerebral blood flow (CBF) and delayed vasospasm was studied in early aneurysm surgery for subarachnoid hemorrhage (SAH). METHODS: Thirty-two patients in this study fulfilled the following criteria: ruptured anterior communicating aneurysms, computed tomography (CT) within 2 days and unilateral pterional approach within 3 days after the ictus, bilaterally symmetrical clots without intracerebral hematoma, no postoperative complication, and CBF studies with single photon emission computed tomography (SPECT) with 123I-IMP. RESULTS: Postoperative regional hypoperfusion due to brain retraction was frequently recognized on 123I-IMP-SPECT without infarction. The regional CBF (rCBF) showed a continuous fall during the first 4 weeks after the ictus, followed by improvement. The rCBF in the vicinity of the surgical route was significantly lower, especially in the acute stage (Day 3-7). A significant association between decrease of cisternal blood after surgery and the degree of local vasospasm and local CBF values during spasm stage was observed in the interhemispheric cisterns, A2 and medial frontal cortex, but not in the sylvian fissure or insular cisterns, M1 or M2, and frontal watershed and temporal cortex. CONCLUSIONS: The present study provides evidence for the effectiveness of direct clot removal by early surgery for SAH on local vasospasm and CBF reduction. However, a potential improvement in local CBF with clot removal could be masked by brain retraction, which was demonstrated to affect rCBF adversely. Therefore, it is critical to perform brain retraction as gently as possible.

Topological mutation of Escherichia coli dihydrofolate reductase.

Proteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, i.e. the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, "topological" mutants (designated as GHF33 and GHF34) of Escherichia coli dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the betaF strand and betaG-betaH strands in the primary sequence. In this way, the chain topology of wild-type DHFR, betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaF-->betaG-->betaH, was changed to betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaG-->betaH-->betaF. Such topological mutant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of kcat, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with Km values of 3.3 microM (GHF33) and 2.1 microM (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with DeltaGH2O values of 4.0 and 4.8 kcal/mol. Whereas, the Km and DeltaGH2O values for wild-type DHFR were 0.9 microM and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.

Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase.

An important consideration in the construction of active and stable circularly permuted proteins is the connective sequence that links the native N- and C-termini. For this reason, various lengths of polyglycine linkers (two, three, four, five and six glycines) were employed to connect the original N- and C-termini of a circularly permuted construct of Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus was Met16. Examination of the circular dichroism (CD) spectra, gel-filtration chromatography elution profiles, urea-induced unfolding properties and enzyme kinetics revealed that, among the linkers tested, a linker length of five glycines was the most favorable. The Vmax of the circularly permuted variant with a five glycine linker (cpM16G5) was about 20% that of wild-type DHFR, although far UV CD spectra, gel filtration elution time, conformational stability and Km for the substrate dihydrofolate and Kd for the coenzyme NADPH were comparable in the two proteins. Another circularly permuted DHFR with a five glycine linker in which a new N-terminus was created at Leu24 (cpL24G5) was also constructed and assayed. The Vmax of cpL24G5 was almost the same as the wild-type, presumably due to the optimization of the glycine linker. The improved activity of the Leu24 permutant is probably due to the disruption of a catalytically important structure, the M20 loop, in the Met16 permutant.

In search of circular permuted variants of Escherichia coli dihydrofolate reductase.

A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate reductase (DHFR) was used to search for a proteolytic site that gave new N- and C-termini on circularized DHFR with enzyme activity. Of the six site-specific proteolytic enzymes tested, three proteases, Achromobacter protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and Staphylococcus aureus V8 protease, cleaved a single site of the circularized DHFR to form circular permuted variants. Twenty-four possible sites for cleavage were found formation of eight circular permuted variants was suggested by results of N-terminal sequence analysis of the linearized proteins isolated by gel filtration in the presence of 5 M guanidine hydrochloride. Mapping of the predicted cleavage sites on the DHFR molecule suggested that they were not all at a specific loop and, therefore, there are many possible circular permuted variants.

Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function.

To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.

Detection of conformational changes in an immobilized protein using surface plasmon resonance.

Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.

Cyanocysteine-mediated molecular dissection of dihydrofolate reductase: occurrence of intra- and inter-molecular reactions forming a peptide bond.

During a cyanocysteine-mediated dissection study of dihydrofolate reductase, a peptide fragment with a molecular mass of 18 Da less than expected was found as a major reaction product when the dissection reaction was applied to a Lys-cyanocysteine linkage. Detailed characterization of the dissection products by protease digestion, peptide sequencing, liquid chromatography/electrospray ionization mass spectrometry, and capillary electrophoresis suggested that the by-product was generated via a lactam ring formation through the intramolecular nucleophilic attack of the epsilon-amino group on the carbonyl carbon of the Lys-cyanocysteine linkage. We have also demonstrated the occurrence of intermolecular attack of an alpha-amino group of glycine on the carbonyl carbon of the X-cyanocysteine linkage to form a new X-Gly linkage, which should be a useful reaction for specific modification of proteins at the C-terminal.

Rotational occlusion of the vertebral artery caused by transverse process hyperrotation and unilateral apophyseal joint subluxation. Case report.

The authors describe transverse process hyperrotation and unilateral apophyseal joint subluxation as a novel mechanism of rotational vertebral artery (VA) occlusion. The patient, a 56-year-old man, complained of episodic bilateral blindness when rotating his head more than 90 degrees to the right. Plain cervical x-ray films showed spondylotic osteophytes of the right C4-5 uncovertebral portion. Dynamic angiography revealed right VA occlusion at C4-5 and left VA occlusion at C1-2 with head rotation to the right. It was demonstrated on three-dimensional images constructed from computerized tomography scans that C-4 transverse process hyperrotation compressed the right VA against the apex of the C-5 subluxating superior articular process via the inner surface of the transverse process anterior root (processus costarius) rather than the osteophytes. It is also proposed that the true site of occlusion is different from that observed in angiographic studies.

Purification and characterization of the flagellar hook-basal body complex of Bacillus subtilis.

The flagellar hook-basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram-negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium. The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B. subtilis FIgG, which was previously reported as a rod protein. The sequence of the reported FIgG protein of B. subtilis is more closely related to that of FIgE (the hook protein) rather than FIgG (the rod protein) of S. typhimurium, in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook-basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N-terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide-gene correspondences: 82 kDa, fIiF; 59 kDa, fIgK; 35 kDa, orfF; 32 kDa, yqhF; 23 kDa, orf3 of the fIaA locus; 20 kDa, fIgB and fIgC; 13 kDa, not determined. The band at 20 kDa was a mixture of FIgB and FIgC, as revealed by two-dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium.

Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

Correlation between measurements using a new halitosis monitor and organoleptic assessment.

Halitosis is known as unpleasant oral odor and is a health concern among the general public. Previously, we reported on a new portable monitor with a zinc-oxide, thin-film, semiconductor sensor which demonstrated simplicity of handling, high reproducibility and correspondence for organoleptic assessment. The results suggested its usefulness for the diagnosis of halitosis. Using the monitor, oral air samples of 94 subjects were measured in a field survey, and the values were compared with the organoleptic rates of corresponding samples assessed by two judges. A highly significant correlation (r = 0.824, P < 0.01) was demonstrated between the measures obtained by the two methods. The results suggest that the monitor is useful for not only a clinical study but also a field study of halitosis.