Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.     

Factors contributing to cerebral hypomyelination in the growth hormone-deficientlittle mouse

We attempted to delineate the events leading to hypomyelination in the brain of thelittle mouse, a promising murine model of isolated growth hormone deficiency. At 20 days of age, the mutant mouse brain weighed less than its normal counterpart, and this difference in brain weight persisted. Increase in CNPase activity was found to be suppressed in the cerebrum throughout the developmental stage, but not in the other parts of the brain. Differences in cerebral DNA content between thelittle and normal mice first became apparent on the 10th day of age. Thereafter, the rate of increase in thelittle brain consistently lagged behind the normal. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the normal cerebrum is most active, was approximately half that of the controls in all parts of thelittle brain. These findings indicate that the hypomyelination of the mutant cerebrum might result from reduced oligodendroglial proliferation due to growth hormone deficiency.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Efficiency of serum copper/zinc ratio for differential diagnosis of patients with and without lung cancer

We examined serum copper (Cu), serum zinc (Zn), and the serum copper/zinc ratio (Cu/Zn) in 162 patients. All of them were seen to have an abnormal shadow in the chest X-ray films, that is, 109 patients with lung cancer (LC) and 53 patients with no lung cancer (NLC). The mean Cu and Cu/Zn in LC patients were significantly higher than those in NLC patients (p<0.05). In LC patients, Cu and Cu/Zn were higher and Zn was lower in advanced tumors than early ones. There was a significantly clear relation between Cu or Cu/Zn and the tumor (T) stages. When the relative risk (RR) of LC was estimated, it was seen that the higher Cu and Cu/Zn became, the higher RR became. Furthermore, we showed the sensitivity of the receiver operator characteristic of the test (ROC) curve for Cu, Cu/Zn, and carcinoembryonic antigen (CEA) to diagnose LC, as explained in a paragraph of methods.The determinations of Cu, Zn, and Cu/Zn are simple and inexpensive. They also appear to have a great diagnostic value in determining the local invasion of LC and as a screening test in the high-risk patients for LC.     

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.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the