Organic-Solvent-Tolerant Bacterium Which Secretes Organic-Solvent-Stable Lipolytic Enzyme

A bacterial strain which could be grown in a medium containing organic solvents and which could secrete lipolytic enzyme was isolated. The stability of the lipolytic activity of the supernatant of the culture increased significantly in the presence of organic solvents such as toluene, cyclohexane, ethanol, and acetone.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Temperature Sensitivity of Agonist High-Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5-fold increase in the agonist high-affinity binding sites of the D₁ dopamine receptor. These high-affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high-affinity values of the receptor for dopamine nor the percent receptors in the high-affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high-affinity D₁ receptors with only 25% of the total receptor population in the high-affinity state; there was no change in the affinity values of the high-affinity binding sites. At 37°C, there was a 40% loss in total number of D₁ receptor binding sites. All the high-affinity binding sites were lost and the remaining 60% of binding activity represented the low-affinity binding state of the receptor. These results indicate that the high-affinity binding sites of the reconstituted D₁ dopamine receptors are uniquely sensitive to higher temperatures.     

Primary culture of rat hepatocytes using membrane-supported collagen sandwich with hormone-free medium

Summary Primary culture of rat hepatocytes in hormone-free medium using membrane-supported collagen sandwich maintained their cellular morphology and expressed albumin secretion for about 3 weeks in vitro. It was reconfirmed that mimicking the cellular environment in vivo was effective for cellular maintenance.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Detection of Antibody-Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections

We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 10⁷ colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 10⁷ colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.