Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts

BACKGROUND: In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. RESULTS: Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha on chromatin. CONCLUSIONS: These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.     

Morphological brain changes associated with Schneider's first-rank symptoms in schizophrenia: a MRI study

BACKGROUND: Schneider's first-rank symptoms involve an alienated feature of the sense of one's own mental or physical activity. To clarify the brain morphological basis for the production of these symptoms, volumes of the frontal and medial temporal regions and their clinical correlates were examined in patients with schizophrenia. METHOD: Twenty-two patients with schizophrenia and 44 age- and gender-matched healthy control subjects were included. All patients were in their psychotic episodes with definite Schneiderian symptoms, rated by using the Scale for Assessment of Positive Symptoms. Volumetric measurements of high-resolution magnetic resonance imaging were performed in the prefrontal area, cingulate gyrus, and precentral gyrus, and the medial temporal structures such as the amygdala, hippocampus, and parahippocampal gyrus. RESULTS: Patients had significantly decreased volumes in the cingulate gray matter and the amygdala compared to controls. In the patient group, Schneiderian symptom severity showed significant inverse correlations with volumes of the right posterior cingulate gray matter and of the left anterior parahippocampal gyrus. CONCLUSIONS: Schneiderian symptoms may be associated with morphological abnormalities in the limbic-paralimbic regions such as the cingulate gyrus and parahippocampal gyrus, which possibly serve the self-monitoring function and the coherent storage and reactivation of information.     

Clinical significance of IgG4 antibody determination in children against egg white, milk, soybean and Dermatophagoides farinae]

The measurement of IgE and IgG4 antibodies against egg white, milk, soybean and Dermatophagoides farinae was performed by FAST (fluorescence allergosorbent test) using 21 serum samples obtained from non-allergic children and 160 serum samples from atopic children with bronchial asthma and/or atopic dermatitis. Their antibody levels were evaluated for any association with disease severity and for clinical significance in establishing diagnosis. It was found that children with bronchial asthma showed lower levels of IgE antibodies against egg white, milk and soybean and higher levels of IgE antibodies against Dermatophagoides farinae compared with those of children with atopic dermatitis, while both groups showed higher levels of egg white and milk-specific IgG4 antibodies compared with non-allergic children. These IgE and IgG4 antibody levels revealed a tendency to correlate with disease severity in patients with atopic dermatitis, while this was not observed in patients with bronchial asthma. The contribution percentages of IgG4 antibody determination, together with IgE antibody determination, in retrieving causal allergens were 71% for egg white, 70% for milk and 48% for soybean allergy, implying their diagnostic value in establishing clinical diagnosis.     

Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis.

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.     

Effects of succinylated concanavalin A on infectivity and syncytial formation of human immunodeficiency virus

The effects of various lectins on the infectivity of human immunodeficiency virus (HIV) type 1 was investigated. Among the 25 lectins investigated, 2 types of concanavalin A (Con A) and 3 types of phytohemagglutinin were found to inhibit HIV infection. Succinylated Con A (S-Con A) efficiently blocked HIV-induced formation of syncytia in a coculture of MOLT-4 cells and blocked cell-free infection by HIV of MT-4 cells. The HIV-binding study revealed that S-Con A only partially inhibited viral binding to cells, although the control Leu-3a monoclonal antibody strongly inhibited it. When S-Con A was added to cultures after the initiation of viral adsorption, the number of HIV antigen-positive cells that developed depended on the time interval before addition of the compound. S-Con A inhibited HIV infection even after viral binding to cells at 0 °C and further incubation at 37 °C for 1 day. These data suggest that S-Con A inhibited mainly the fusion process rather than viral binding to cells in exerting its anti-HIV activity.     

Splanchnic clearance of plasma vasopressin in the dog: evidence for prehepatic extraction

It is generally considered that the liver is primarily responsible for the extraction of vasopressin from the circulating blood by the splanchnic viscera. To investigate this matter further, measurements were made in the anesthetized dog of the concentrations of vasopressin in arterial, portal venous, and hepatic venous plasma, and of total splanchnic plasma flow and hepatic arterial plasma flow. The total splanchnic vasopressin extraction ratio was 12.9 +/- 1.0%. However, the concentration of vasopressin in portal venous plasma was consistently lower than in arterial plasma, and there was a substantial prehepatic extraction of vasopressin, averaging 10.5 +/- 0.8%. A quantitative evaluation of the contribution of the "prehepatic" viscera, i.e., viscera with venous drainage into the portal vein, is provided by the relevant clearances of vasopressin. The prehepatic and total splanchnic vasopressin clearances were 1.58 +/- 0.20 and 3.04 +/- 0.31 ml X min-1 X kg-1, respectively. Thus, the splanchnic viscera other than the liver were responsible for approximately half of the splanchnic clearance of vasopressin; the remainder could be attributed to the liver. Immunoreactive vasopressin was not found in the bile. In splenectomized dogs, in which venous blood was collected from the superior mesenteric vein, the vasopressin extraction ratio was 14.6 +/- 2.3%, suggesting that the prehepatic clearance of vasopressin occurs largely in the mesenteric bed. A more specific localization of the prehepatic clearance sites has not as yet been made.     

Cartilage macromolecules and the calcification of cartilage matrix

The calcification of cartilage matrix in endochondral bone formation occurs in an extracellular matrix composed of fibrils of type II collagen with which type X collagen is closely associated. Also present within this matrix are the large proteoglycans containing chondroitin sulfate which aggregate with hyaluronic acid. In addition, the matrix contains matrix vesicles containing alkaline phosphatase. There is probably a concentration of calcium as a result of its binding to the many chondroitin sulfate chains. At the time of calcification, these proteoglycans become focally concentrated in sites where mineral is deposited. This would result in an even greater focal concentration of calcium. Release of inorganic phosphate, as a result of the activity of alkaline phosphatase, can lead to the displacement of proteoglycan bound calcium and its precipitation. The C-propeptide of type II collagen becomes concentrated in the mineralizing sites, prior to which it is mainly associated with type II collagen fibrils and is present in dilated cisternae of the enlarged hypertrophic chondrocytes. The synthesis of type II collagen and the C-propeptide, together with alkaline phosphatase, are regulated by the vitamin D metabolites 24,25(OH)2 cholecalciferol and 1,25 (OH)2 cholecalciferol. At the time of calcification, type X collagen remains associated with type II collagen fibrils. It may play a role in preventing the initial calcification of these fibrils focusing mineral formation in focal interfibrillar sites. This process of calcification is clearly very complex, and involves different interacting matrix molecules and is carefully regulated at the cellular level.     

M-CSF levels during peripheral blood stem cell harvest and autologous stem cell transplantation

Plasma macrophage colony-stimulating factor(M-CSF) levels were determined during peripheral blood stem cell harvest and autologous stem cell transplantation(PBSCT). Plasma of 10 patients were analyzed by using ELISA system. The average peak values during PBSCT were quite higher than those during harvest(20,092 vs 1,681 pg/ml). Peak values were observed mainly around leukocyte nadir(Phase II) during harvest. On the other hand, they were detected just after pretreatment(Phase I) during PBSCT courses. Moreover, samples showing extremely high M-CSF values(Phase I) were associated with increase in serum LDH levels. These data suggest that plasma M-CSF in Phase I are mainly derived from chemotherapy-induced cellular damage during PBSCT courses, and there might be different mechanisms to raise M-CSF around the nadir of leukocytes. It is necessary to elucidate the biological meanings of M-CSF.     

Genetic control in the susceptibility of germfree inbred mice to infection by Escherichia coli O115a,c:K(B).

We studied the susceptibility of five germfree inbred strains of mice to oral infection by murine pathogenic Escherichia coli O115a,c:K(B) (MPEC), the causative agent of mouse megaenteron. Although MPEC colonized all strains of mice at 10(9)/g of feces, the mouse strains could be divided into three groups according to their intestinal lesions. In CF1 and C3H/He mice, intestinal lesions were produced in the cecum and colon with hyperplasia of epithelial cells accompanied by severe inflammatory reactions and erosion. The lesions in NC and C57BL/6 mice were restricted to the tip of the cecum, and hyperplasia of epithelial cells was more severe in these mice than in CF1 or C3H/He mice. BALB/c mice had no lesions. Analysis of F1 hybrids of CF1, NC, and BALB/c mice and offsprings from backcrosses of F1 mice to parental strains showed that susceptibility to MPEC seemed to be controlled genetically by a single locus which may be related to the receptors on epithelial cells for MPEC adherence. However, the differences in lesions between CF1 and NC mice suggest that a combination of this locus and another locus to which it may be related regulates the hyperplasia of intestinal epithelial cells.