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.     

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.     

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.     

The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus.

The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.     

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     

Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the -amylases. The optimum pH, specific activity and K _m value for -cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mm, respectively. These values were almost identical to those from B. sphaericus E-244. Correspondence to: T. Oguma