The bronchial tree,lobular division,and blood vessels of the lung of the silvered lutong (Presbytis cristata)

The lungs of three silvered lutongs (Presbytis cristata) were examined. The right and left lungs have the dorsal, lateral, ventral, and medial bronchiole systems, which arise from the corresponding sides of both bronchi, respectively. Bronchioles in the dorsal and lateral bronchiole systems are well developed, whereas those in the ventral and medial bronchiole systems are poorly developed and lack some portions. According to the fundamental structure of bronchial ramifications of the mammalian lung (Nakakuki, 1975, 1980), the right lung consists of the upper, middle, lower, and accessory lobes, whereas the left lung consists of a bilobed middle lobe and a lower lobe, in which the right upper lobe is extremely well developed. The right pulmonary artery runs across the ventral side of the right upper lobe bronchiole, and then across the dorsal side of the right middle lobe bronchiole. Initially it runs along the lateral side of the right bronchus and then gradually comes to run along the dorsal side. During its course, it gives off branches which run mainly along the dorsal or lateral side of the bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, and then follows the same course as that in the right lower lobe. The pulmonary veins run medially or ventrally to the bronchioles, and finally enter the left atrium as four or five large veins.     

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.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Effects of vasoactive intestinal peptide on glycogenolysis in cultured liver cells.

When isolated rat liver cells were incubated in the presence of vasoactive intestinal peptide at the concentrations ranging from 0.2 microgram to 2 micrograms per ml, glycogenolysis was maximally stimulated within 15 min. However, somatostatin inhibited the liver glycogenolysis. The combined addition to the incubation medium showed that insulin and somatostatin inhibited the stimulated glycogenolysis induced by vasoactive intestinal peptide, while vasoactive intestinal peptide plus secretin showed no additive effect on glycogenolysis, as compared with single the addition of vasoactive intestinal peptide. On the other hand, the additon of glucagon to vasoactive intestinal peptide showed additive effects on glycogenolysis. These results suggest that the receptor site for vasoactive intestinal peptide may be distinguishable from that for glucagon. Extracellular calcium ions were demonstrated to play an important role in the modulation of vasoactive intestinal peptide-induced glycogenolysis. The evidence presented in this paper indicates that glucose metabolism may be partly regulated by the direct action of vasoactive intestinal peptide on hepatocytes, which is referred to as an enterohepatic axis and that the axis is inhibited by insulin and somatostatin.     

Kinetics of proton transfer reactions of lysozyme with p-nitrophenol near neutral pH—a study of dynamic properties of Glu 35 and His 15

Kinetics of proton transfer between lysozyme and a pH indicator p-nitrophenol (p-Np) were measured by the temperature-jump method in a pH range of 6.0–7.0. Two well-defined relaxation processes were observed. The fast process (τ ? 15 μsec) was also observed for a lysozyme derivative succinylated at the terminal α-amino group of Lys 1. Therefore, the fast process was found to be attributable to the proton transfer reaction of His 15 with p-Np. The slow process (τ ? 50 μsec) was found to be characteristic of the proton transfer reaction of Glu 35, because it disappeared completely in solution containing a lysozyme derivative having an ester crosslink between the carboxyl group of Glu 35 and indol C-2 of Trp 108. The rate constants for proton transfer from Glu 35 and His 15 to p-Np were found to be 9 × 10⁶/sec/M (±65%, 23°C) and 3 × 10⁸/sec/M (±20%, 25°C), respectively. These data indicate that the proton of the carboxyl group of Glu 35 is kinetically stabilized in lysozyme.     

ABCG2/BCRP Dysfunction as a Major Cause of Gout

Recent genome-wide association studies showed that serum uric acid (SUA) levels relate to ABCG2/BCRP gene, which locates in a gout-susceptibility locus revealed by a genome-wide linkage study. Together with the ABCG2 characteristics, we hypothesized that ABCG2 transports urate and its dysfunction causes hyperuricemia and gout. Transport assays showed ATP-dependent transport of urate via ABCG2. Kinetic analysis revealed that ABCG2 mediates high-capacity transport of urate (Km: 8.24 ± 1.44 mM) even under high-urate conditions. Mutation analysis of ABCG2 in 90 Japanese hyperuricemia patients detected six nonsynonymous mutations, including five dysfunctional variants. Two relatively frequent dysfunctional variants, Q126X and Q141K, were then examined. Quantitative trait locus analysis of 739 Japanese individuals showed that Q141K increased SUA as the number of minor alleles of Q141K increased (p = 6.60 × 10^?5). Haplotype frequency analysis revealed that there is no simultaneous presence of Q126X and Q141K in one haplotype. Becuase Q126X and Q141K are assigned to nonfunctional and half-functional haplotypes, respectively, their genotype combinations are divided into four functional groups. The association study with 161 male gout patients and 865 male controls showed that all of those with dysfunctional ABCG2 increased the gout risk, especially those with ≤1/4 function (OR, 25.8; 95% CI, 10.3–64.6; p = 3.39 × 10^?21). These genotypes were found in 10.1% of gout patients, but in only 0.9% of control. Our function-based clinicogenetic (FBCG) analysis showed that combinations of the two dysfunctional variants are major causes of gout, thereby providing a new approach for prevention and treatment of the gout high-risk population.