Glycated albumin but not HbA1c reflects glycaemic control in patients with neonatal diabetes mellitus.

AIMS/HYPOTHESIS: It is difficult to use HbA(1c) as an indicator of glycaemic control in patients with neonatal diabetes mellitus (NDM) because of high levels of fetal haemoglobin (HbF) remaining in the blood. In this study, glycated albumin (GA), which is not affected by HbF, and HbA(1c) were compared to evaluate whether they reflect glycaemic control in patients with NDM. METHODS: This study included five patients with NDM. Age at diagnosis was 38 ± 20 days. Insulin therapy was started in all patients, and levels of GA, HbA(1c) and HbF were measured monthly for 6 months. One-month average preprandial plasma glucose (aPPG) was calculated using self-monitoring of blood glucose. RESULTS: Plasma glucose and GA were elevated (29.7 ± 13.1 mmol/l [n = 5] and 33.3 ± 6.9% [n = 3], respectively) but HbA(1c) was within normal limits (5.4 ± 2.6% [35.5 ± 4.9 mmol/mol]; n = 4) at diagnosis. With diabetes treatment, aPPG (r = -0.565, p = 0.002), GA (r = -0.552, p = 0.003) and HbF (r = -0.855, p < 0.0001) decreased with age, whereas HbA(1c) increased (r = 0.449, p = 0.004). GA was strongly positively correlated with aPPG (r = 0.784, p < 0.0001), while HbA(1c) showed no correlation with aPPG (r = 0.221, p = 0.257) and was significantly inversely correlated with HbF (r = -0.539, p = 0.004). CONCLUSIONS/INTERPRETATION: GA is a useful indicator of glycaemic control in patients with NDM, whereas HbA(1c) is influenced by age-related changes in HbF and does not accurately reflect glycaemic control.

Crosstalk of prolyl isomerases, Pin1/Ess1, and cyclophilin A.

Previous studies have indicated that Ess1/Pin1, a gene in the parvulin family of peptidyl-prolyl isomerases (PPIases), plays an important role in regulating the G(2)/M transition of the cell cycle by binding cell-cycle-regulating proteins in eukaryotic cells. Although the ess1 gene has been considered to be essential in yeast, we have isolated viable ess1 deletion mutants and demonstrated, via analysis of yeast gene expression profiles using microarray techniques, a novel regulatory role for ESS1 in the G(1) phase. Although the overall expression profiles in the tested strains (C110-1, W303, S288c, and RAY-3AD) were similar, marked changes were detected for a number of genes involved in the molecular action of ESS1. Among these, the expression levels of a cyclophilin A gene, also a member of the PPIase family, increased in the ess1 null mutant derived from C110-1. Subsequent treatment with cyclosporin A significantly retarded growth, which suggests that ESS1 and cyclophilin A are functionally linked in yeast cells and play important roles at the G(1) phase of the cell cycle.

Two mutations of the Gsalpha gene in two Japanese patients with sporadic pseudohypoparathyroidism type Ia.

Pseudohypoparathyroidism Ia (PHP-Ia), is an inherited disease with clinical hypoparathyroidism caused by parathyroid hormone resistance (PTH), and shows the phenotype of Albright hereditary osteodystrophy (AHO), including short stature, obesity, round face, brachydactyly, and subcutaneous ossification. This disease is caused by mutation that inactivates the alpha-subunit of Gs, the stimulatory regulator of adenylyl cyclase. Here, a novel frameshift mutation (delG at codon 88) in exon 4, and a missense mutation (R231H) in exon 9 of the Gsalpha gene were identified in two Japanese patients with sporadic PHP-Ia. Deletion of a G in exon 4 at codon 88 in the first patient produced a premature stop codon, resulting in the truncated protein. The second patient had a previously reported R231H mutation. Because this amino acid is located in a region, switch 2, that is thought to interact with the betagamma subunit of Gsalpha protein, this mutation may impair Gs protein function. We report here one novel Gsalpha mutation, and note that mutations in Japanese patients with PHP-Ia are probably heterogeneous.

Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is an X-linked dominant disorder characterized by renal phosphate wasting, abnormal vitamin D metabolism, and defects of bone mineralization. The phosphate-regulating gene on the X-chromosome (PHEX) that is defective in XLH has been cloned, and its location identified at Xp22.1. It has been recognized to be homologous to certain endopeptidases. So far, a variety of PHEX mutations have been identified mainly in European and North American patients with XLH. To analyze the molecular basis of four unrelated Japanese families with XLH, we determined the nucleotide sequence of the PHEX gene of affected members. We detected a new nonsense mutation (R198X) in exon 5, a new 3 nucleotides insertion mutation in exon 12 and a new missense mutation (L160R) in exon 5 as well as a previously reported nonsense mutation in exon 8 (R291X). These results suggest that: 1) PHEX gene mutations are responsible for XLH in Japanese patients, and 2) PHEX gene mutations are heterogeneous in the Japanese population similarly to other ethnic populations.

Older boys benefit from higher initial prednisolone therapy for nephrotic syndrome. The West Japan Cooperative Study of Kidney Disease in Children.

BACKGROUND: A long course of the initial prednisolone therapy has been shown to be more effective than standard-course therapy in reducing relapse rates in children with idiopathic nephrotic syndrome, but it is commonly accompanied by corticosteroid toxicities. There has been no study on prednisolone dosage for the effective treatment of nephrotic syndrome. METHODS: Sixty-eight children (42 boys and 26 girls) with an initial attack of nephrotic syndrome were randomly allocated into two different long-course treatment groups. Patients in Group 1 received a daily prednisolone dose of 60 mg/m2 for six weeks, followed by an alternate-day dose of 40 mg/m2 for six weeks. Patients in Group 2 had a daily dose of 40 mg/m2 instead of 60 mg/m2. RESULTS: Four children in each group did not respond within six weeks. Group 1 was associated with a significantly earlier response but more frequent corticosteroid toxicities than Group 2. Boys in Group 1 had a higher rate of sustained remission than boys in Group 2 (P = 0.0073), especially boys four years old or more (P = 0.0027), but girls did not show a significant difference (P = 0.863). Boys four years old or more in Group 1 had a course of frequent relapsing less often than those in Group 2 (2 of 13 vs. 6 of 8, P = 0.0075). CONCLUSION: These findings indicate that efficient prednisolone doses may vary between sexes and ages, and that a higher initial prednisolone therapy may be of greater benefit to older boys.

Gonadotropin-releasing hormone analog therapy failed to improve predicted final height in two children with central precocious puberty and microcephalus.

Long-acting gonadotropin-releasing hormone (GnRH) analog treatment for central precocious puberty (CPP) suppresses excessive bone maturation by inhibiting the pituitary-gonadal axis, and usually assures favorable results for growth potential. Recently, we encountered two children with CPP and microcephalus in whom GnRH analog therapy arrested pubertal development, but could not suppress bone age maturation effectively. Eventually, their final height deteriorated rather than improved. The reason why these two cases did not respond to GnRH analog therapy remains unknown. However, microcephalus and minor cerebral anomalies may have some links to deterioration of final height. Our cases suggest that careful evaluation will be required especially for CPP with microcephalus throughout treatment with GnRH analog.

A novel missense mutation in the HMG box region of the SRY gene in a Japanese patient with an XY sex reversal.

The sex-determining region of the Y chromosome, the SRY gene, located on the short arm of the Y chromosome, is appreciated as one of the genes that is responsible for directing the process of sex differentiation. To date, 34 different mutations, including 29 missense and nonsense mutations in the SRY gene, have been described in XY female patients. We investigated the molecular basis of the sex reversal in one Japanese XY female patient by determining the nucleotide sequence of the SRY gene, using polymerase chain reaction and direct sequencing. We identified a novel mutation, of the substitution of Tyr for Asn at nucleotide position 87 (N87Y). This Asn residue is located within the DNA-binding high-mobility-group (HMG) motif, which is considered to be the main functional domain of the SRY protein. Further, this amino acid, Asn, is a conserved residue among mammalian SRY genes. These findings indicate that this amino acid substitution may be responsible for the sex reversal in this patient.

p97 ATPase, an ATPase involved in membrane fusion, interacts with DNA unwinding factor (DUF) that functions in DNA replication.

DNA unwinding factor (DUF) unwinds duplex DNA and is supposed to function in DNA replication in Xenopus egg extracts. Here we report the isolation and analysis of a DUF-interacting factor. By immunoprecipitation, we found that p97 ATPase (p97) interacts with DUF in Xenopus egg extracts. This interaction was confirmed by the in vitro binding of purified p97 with DUF. When sperm chromatin was added to Xenopus egg extracts to construct nuclei active in DNA replication, p97 was incorporated into the nuclei. These data suggest that the complex of DUF and p97 may function in DNA replication.

Learning to design synergetic computers with an extended symmetric diffusion network.

This article proposes an extended symmetric diffusion network that is applied to the design of synergetic computers. The state of a synergetic computer is translated to that of order parameters whose dynamics is described by a stochastic differential equation. The order parameter converges to the Boltzmann distribution, under some condition on the drift term, derived by the Fokker-Planck equation. The network can learn the dynamics of the order parameters from a nonlinear potential. This property is necessary to design the coefficient values of the synergetic computer. We propose a searching function for the image processing executed by the synergetic computer. It is shown that the image processing with the searching function is superior to the usual image-associative function of synergetic computation. The proposed network can be related, as a special case, to the discrete-state Boltzmann machine by some transformation. Finally, the extended symmetric diffusion network is applied to the estimation problem of an entire density function, as well as the proposed searching function for the image processing.

A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs.

BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

A Japanese case with Frasier syndrome caused by the splice junction mutation of WT1 gene.

The Wilms' tumor suppressor gene, WT1, plays an important role in the development of the urogenital system and also subsequent normal function of this system. Recently, the splice mutations in intron 9 of WT1 gene have been detected in Frasier syndrome, which is characterized by streak gonads, pseudohermaphroditism, slowly progressive nephropathy and frequent development of gonadoblastoma. Here to elucidate the molecular basis in a Japanese patient of Frasier syndrome, WT1 gene was analyzed by polymerase-chain-reaction (PCR) and direct sequencing. We identified the splice junction mutation in intron 9 of WT1, which is recognized as a mutation hot-spot in intron 9. This finding concludes that 1) the mutation in intron 9 might be the cause of Frasier syndrome, and 2) the mutation hot-spot in Japanese and Caucasian patients is similar.

Modulation of DNA synthesis by microtubule-associated protein 2 in the nuclear matrix isolated from Physarum polycephalum.

The mechanism of stimulation of DNA synthesis by microtubule-associated protein 2 (MAP2) was examined in the nuclear matrix isolated from Physarum polycephalum. Porcine brain MAP2 stimulated DNA synthesis by the matrix with exogenous templates, but not with endogenous templates. Kinetic analyses showed that MAP2 decreases the Km of the matrix for deoxyribonucleoside triphosphates. Comparison of the Km values of active- and latent-type DNA replication machineries of Physarum suggested a possible role for MAPs or MAP-like proteins in DNA replication.

Initiation of DNA synthesis in a high molecular weight fraction of Xenopus egg extract.

Xenopus egg extract was fractionated by gel-filtration column chromatography and DNA synthetic activity was examined using double-stranded DNA as a template. The major activity eluted had an apparent molecular mass of about 300 kDa. Product analyses showed that de novo DNA synthesis occurs with formation of replication bubbles, thereby suggesting that this fraction catalyzes the initiation of DNA replication. Activities of DNA polymerase alpha-primase and DNA helicase overlapped with the DNA synthetic activity, but the elution profiles of the enzymes differed from that of the DNA synthetic activity. Therefore, this 300-kDa fraction may contain a component which differs from the above enzymes yet is essential for initiation of DNA replication.

Stimulation of DNA polymerase alpha activity by microtubule-associated proteins.

Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.

Microtubule destabilization by cdc2/H1 histone kinase: phosphorylation of a "pro-rich region" in the microtubule-binding domain of MAP-4.

Microtubule-associated protein-4 (MAP-4), a major MAP in proliferating cells, consists of a microtubule-binding domain and a projection domain protruding from the microtubule wall. The former contains a Pro-rich region and an assembly-promoting (AP) sequence region which is common to the neuron-specific MAPs, MAP-2 and tau1. In this paper, we describe the phosphorylation of the Pro-rich region of MAP-4 and the suppression of its assembly-promoting activity by cdc2/H1 histone kinase. This inactivation of MAP-4 may cause disassembly of the interphase microtubular network at the end of the G2 phase of the cell cycle.

The molecular structure of adrenal medulla kinesin.

The molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low-angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low-ionic solution, and the ratio of extended molecules increased to 40-50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod-shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two-headed structure of myosin, while the other end had a more ill-defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan-like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP-PNP) also displayed a folded morphology.

Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization.

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity-purified antibody against bovine adrenal kinesin. It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody-stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine-phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold-treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37 degrees C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.

Purification and characterization of kinesin from bovine adrenal medulla.

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.

Anti-streptococcal cell membrane and anti-human glomerular basement membrane titers in sera of patients with poststreptococcal acute glomerulonephritis and anaphylactoid purpura.

To clarify the immunological mechanisms in poststreptococcal acute glomeulonephritis (PSAGN) and anaphylactoid purpura (AP), anti-streptococcal cell membrane (anti-SCM) and anti-human glomerular basement membrane (anti-GBM) titers in the sera of patients with PSAGN and AP were determined by passive hemagglutination with chromic chloride-treated sheep erythrocytes. Sodium lauryl sulfate (SLS) soluble SCM and collagenase soluble GBM were used as soluble antigens. Positive anti-SCM titers (greater than 1:8) were demonstrated in 10 of 14 patients (71.4%) with PSAGN and in 4 of 9 patients (44.4%) with AP with evidence of antecedent streptococcal infection. Two of 4 patients with AP without evidence of antecedent streptococcal infection had positive anti-SCM titers. No correlation was noted between anti-streptolysin O (ASO) titers and anti-SCM titers in patients with PSAGN or AP, but many patients with high ASO titers also had high anti-SCM titers. No positive anti-GBM reactions were detected in patients with PSAGN or AP. No cross-reactions were noted between SLS soluble SCM and collagenase soluble GBM.