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Biomedical subjects

K Kuwajima

Publications and source records attributed to K Kuwajima.

At least 73 records · Page 4Linked to original sources
PubMed1992

Hyper-response of serum IgG1 to Staphylococcus aureus peptidoglycan in patients with hyper-IgE syndrome.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis and recurrent infections. To determine whether an impairment of the antibody response to Staphylococcus aureus contributes to infections in this syndrome we measured total serum IgG subclass, specific IgG1 and IgG2 levels against peptidoglycan (PG), the immunodominant cell wall component of S. aureus and serum opsonic activity to PG. Of the 14 patients with HIE syndrome, nine had increased level of serum IgG1 and six had IgG2 subclass deficiency. In regard to specific response of IgG1 and IgG2 antibodies to PG, patients were divided into five groups related to ages and compared with 10 control subjects for each age cohort. Patients with HIE syndrome had significant high levels of serum-specific IgG1 to PG and significant decreased levels of serum-specific IgG2 to PG in all five groups. Additionally, serum opsonic activity in patients was significantly higher than that in normal control subjects. It is concluded that IgG2 deficiency or poor IgG2 antibody response to S. aureus is not the explanation of the abnormal susceptibility to S. aureus infections of HIE patients.

Adolescent↗
PubMed1992

Familial occurrence of ring chromosome 15.

We present a family with the ring 15 chromosome (r(15)) syndrome in a 2-year-old infant and his mother. Both had the common clinical manifestations reported in previous cases with r(15), including severe short stature, microcephaly, triangular face, and mild mental retardation. The family also had a normal son. Although males with r(15) syndrome usually seem to be infertile, female r(15) patients are likely to be fertile and their reproductivity may be influenced by severe growth retardation.

Adult↗
PubMed1992

[Reducing body myopathy--a case report].

A 2-year-old girl with reducing body myopathy was reported. She had no family history of neuromuscular disease. She developed normally with no delay in milestones during infancy. She had no muscle weakness or hypotonia up to 2 years of age when she received mumps vaccination. Three days after the injection, she was first noticed to have limb muscle weakness. The muscle weakness progressed rapidly with increasing difficulty in gait and raising the upper arms, particularly the left. Four months later, she had difficulty in keeping her head up and could no longer climb the stairs. On physical examination, she had proximal dominant generalized muscle weakness, with a preferential neck muscle involvement. She walked waddlingly and stood up with Gowers' maneuver. Facial and ocular muscles were intact. No dysarthria, dysphagia or respiratory difficulty was noted. EMG showed myopathic pattern. Serum creatine kinase level was moderately elevated to 739 IU/l. In the biopsied left biceps muscle, there was marked variation in fiber size, but no apparent necrotic or regenerating fibers. The most striking feature was the presence of numerous eosinophilic inclusions which reduced nitroblue tetrazolium (NBT) and were, therefore, stained dark with menadione-linked alpha-glycerophosphate dehydrogenase even without the substrate of menadione, showing the histochemical characteristics of "reducing" body. The bodies were predominantly seen in fibers with disorganized intermyofibrillar networks and with high acid phosphatase activity. On electron microscopy, the reducing bodies consisted of fine granular material with the similar electron density to the chromatin granules and were located mostly around the degenerated nuclei, suggesting the nuclear degeneration playing a role in forming the reducing bodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Child, Preschool↗
PubMed1991

Weaver syndrome in two Japanese children.

We report on 2 Japanese patients (a 3-year-old girl and an 20-month-old boy) with the Weaver syndrome. The clinical manifestations are mild mental retardation, overgrowth with accelerated bone age, minor facial anomalies including broad forehead, mild hypertelorism, depressed nasal bridge, accentuated philtrum, micrognathia and large ears, and unique behavior characteristics with some social withdrawal. The nosology of the Weaver and Simpson-Golabi-Behmel syndromes is discussed.

Abnormalities, Multiple↗
PubMed1991

Diagnostic hand anomalies in Smith-Magenis syndrome: four new patients with del (17)(p11.2p11.2)

We report clinical and cytogenetic findings of 4 children (2 boys and 2 girls) with the Smith-Magenis syndrome. All 4 patients had an interstitial deletion of 17p: del(17) (p11.2p11.2). Their clinical manifestations included brachycephaly, midface hypoplasia, prognathism, upper lip eversion, short and broad hands with short fingers, clinodactyly of the fifth fingers, fingertip pads, moderate mental retardation, and behavior problems. Analysis of the metacarpophalangeal pattern profiles in patient 2 showed progressive shortness from the metacarpals to the proximal, middle, and the distal phalanges. The fingerpads observed in all 4 patients have hitherto been noted in only one of 26 previously reported patients with the syndrome. These findings serve as a useful clue to the diagnosis of the syndrome.

Abnormalities, Multiple↗
PubMed1991

The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding-folding kinetics.

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro117 is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.

Bacterial Proteins↗
PubMed1991

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy.

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗
PubMed1991

Folding of staphylococcal nuclease A studied by equilibrium and kinetic circular dichroism spectra.

The urea-induced unfolding of staphylococcal nuclease A has been studied by circular dichroism both at equilibrium and by the kinetics of unfolding and refolding (pH 7.0 and 4.5 degrees C), as a function of Ca2+ and thymidine 3',5'-diphosphate (pdTp) concentration. The results are as follows. (1) The unfolding transition is shifted to higher concentrations of urea by Ca2+ and pdTp, and the presence of both ligands further stabilizes the protein. (2) In the first stage of kinetic refolding, the peptide ellipticity changes rapidly within the dead time of stopped-flow measurement (15 ms), indicating accumulation of a transient intermediate. This intermediate is remarkably less stable than those of other globular proteins previously studied. (3) Dependence of the folding and unfolding rate constants on urea concentration indicates that the critical activated state of folding ("transition state") has considerable structural organization. The transition state does not, however, have the capacity to bind Ca2+ and pdTp, as indicated by the effects of these ligands on the unfolding rate constant. (4) There are at least four different phases in the refolding kinetics in native conditions below 1 M urea. In the absence of pdTp, there are two phases in unfolding, while in the presence of pdTp the unfolding kinetics show a single phase. Some characteristics of the transient intermediate and of the transition state for folding are discussed.

Binding Sites↗
PubMed1991

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin.

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.

Binding Sites↗
PubMed1990

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond.

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.

Chemical Phenomena↗
PubMed1990

An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.

The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.

Circular Dichroism↗
PubMed1990

A Japanese male infant with the Weaver syndrome.

A 15-month-old male infant who had pre- and postnatal overgrowth, accelerated bone maturation and characteristic facial appearance was described. Although a Japanese female with Weaver syndrome previously reported had slightly different clinical manifestations from others, our patient had typical clinical features of Weaver syndrome. We suggest that a genetic mutation of the syndrome may be the same in Japanese as other ethnic groups and that Weaver syndrome may be an autosomal dominant disorder with variable expressions.

Bone Diseases, Developmental↗
PubMed1989

Characterization of the critical state in protein folding. Effects of guanidine hydrochloride and specific Ca2+ binding on the folding kinetics of alpha-lactalbumin.

The reversible unfolding and refolding kinetics of alpha-lactalbumin induced by concentration jump of guanidine hydrochloride were measured at pH 7.0 and 25 degrees C using tryptophan absorption at 292 nm, with varying concentrations of the denaturant and free Ca2+. The refolding reaction of alpha-lactalbumin from the fully unfolded (D) state occurs through the two stages: (1) instantaneous formation of a compact intermediate (the A state) that has a native-like secondary structure; (2) tight packing of the preformed secondary structure segments to lead finally to the native structure, this stage being the rate-determining step of the reaction and associated with acquisition of the specific structure necessary for strong Ca2+ binding. Under strongly native conditions, the observed kinetics of refolding is also complicated by the presence of a slow-folding species (10%) in the unfolded state. Considering these facts, the microscopic rate constants in folding and unfolding directions have been evaluated from the observed kinetics and from the equilibrium constants of the transitions among the native (N), A and D states. Close linear relationships have been found in the plots of the activation free energies, obtained from the microscopic rate constants, against the denaturant concentration. They are similar to the linear relationship between the free energy of unfolding and the denaturant concentration. It was demonstrated that the slope of the plots should be approximately proportional to a change in accessible surface area of the protein during the respective activation process, and that only a third of the difference in accessible surface area between A and N is buried in the critical activated state of folding. However, the selective effect of Ca2+ binding on the folding rate constant has been observed also, demonstrating that the specific Ca2+-binding substructure in the N state is already organized in the activated state. Thus, only a part of the protein molecule involving the Ca2+-binding region is organized in the activated state, with the other part of the molecule being left less organized, suggesting that the second stage of folding may be a sequential growing process of organized assemblage of the performed secondary structure segments.

Animals↗
PubMed1988

Insulin binding to circulating monocytes in children with insulin-dependent and non-insulin-dependent diabetes mellitus.

This study describes insulin binding to circulating monocytes in 24 children with insulin-dependent diabetes mellitus (IDDM), five children with non-insulin-dependent diabetes mellitus (NIDDM), and 10 healthy and 12 obese control children. Insulin binding to monocytes was greatly increased in untreated IDDM children with obvious ketoacidosis (5.51 +/- 3.49 vs. 1.91 +/- 0.47 pg/10(6) cells, P less than 0.01), whereas it was decreased in those without obvious ketoacidosis (1.39 +/- 0.30 vs. 1.91 +/- 0.47 pg/10(6) cells, P less than 0.01). Insulin treatment restored insulin binding almost to the level of control children in both ketoacidotic and non-ketoacidotic patients. Insulin binding to monocytes was markedly decreased in untreated NIDDM children with hyperinsulinemia compared with healthy control children (0.73 +/- 0.27 vs. 1.91 +/- 0.47 pg/10(6) cells, P less than 0.01) or obese control children (0.73 +/- 0.27 vs. 1.33 +/- 0.35 pg/10(6) cells, P less than 0.01). These data indicate that changes in insulin secretion and metabolic conditions might be involved in the fluctuation of the number of insulin receptors in IDDM children as well as in NIDDM children.

Adolescent↗
PubMed1987

Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism.

Kinetic refolding reactions of ferricytochrome c and beta-lactoglobulin have been studied by stopped-flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side-chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped-flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.

Circular Dichroism↗