HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway during Induction of Photosynthesis in Maize Leaves

Changes in the level of metabolites of the C₄ cycle and reductive pentose phosphate (RPP) pathway were measured simultaneously with induction of photosynthesis in maize (Zea mays L.) to evaluate what may limit carbon assimilation during induction in a C₄ plant.

After 20 minutes in the dark, there was an immediate rise in photosynthesis during the first 30 seconds of illumination, followed by a gradual rise approaching steady-state rate after 20 minutes of illumination. Among metabolites of the C₄ cycle, there was a net increase in the level of C₃ compounds (the sum of pyruvate, alanine, and phosphoenolpyruvate) during the first 30 seconds of illumination, while there was a net decrease in the level of C₄ acids (malate plus aspartate). The total level of metabolites of the C₄ cycle underwent a sharp increase during this period. At the same time, there was a sharp rise in the level of intermediates of the RPP pathway (ribulose-1,5-bis-phosphate, 3-phosphoglycerate, dihydroxyacetonephosphate, and fructose-1,6-bisphosphate) during the first minute of illumination. The net increase of carbon among intermediates of the C₄ cycle and RPP pathway was far above that of carbon input from CO₂ fixation, and the increase in intermediates of the RPP pathway could not be accounted for by decarboxylation of C₄ acids, suggesting that an endogenous source of carbon supplies the cycles. After 3 minutes of illumination there was a gradual rise in the levels of intermediates of the C₄ cycle and in the total level of metabolites measured in the RPP pathway. This rise in metabolite levels occurs as photosynthesis gradually increases and may be required for carbon assimilation to reach maximum rates in C₄ plants. This latter stage of inductive autocatalysis through the RPP pathway may contribute to the final buildup of these intermediates.

     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching

We have characterized circular DNA in mouse splenocytes treated with the mitogen lipopolysaccharide (LPS) and various cytokines, including transforming growth factor beta (TGF-beta) and interleukin 4 (IL-4). Using probes of immunoglobulin heavy chain constant genes (CH), excision products of class switch recombination were identified. The majority of the clones contained the 3' portion of the switch mu (S mu) region and the 5' portion of other switch regions. Some clones contained 3'-S gamma sequences instead of 3'-S mu. This indicates that isotype switching may occur not only from C mu, but also from one of the C gamma genes to other CH genes further down-stream. In the presence of LPS, the cytokine TGF-beta enhanced the detection of 5'-S alpha-positive clones, while the lymphokine IL-4 enhanced 5'-S gamma 1 positives. The data support the notion that TGF-beta and IL-4 can direct isotype-specific class switching.     

Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activation by Inorganic Phosphate through Stimulating the Binding of the Activator CO2 to the Activation Sites

The mechanism of the regulation of the activation of ribulose1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) by inorganicphosphate (P_i) in the presence of limiting concentrations ofCO₂ was explored. The activation state of RuBisCO increasedsigmoidally following a biphasic kinetics against the concentrationof P_i in the activation mixture with an intermediary plateauat 2 to 3 mM P_i when the enzyme was activated for 30 min. Theintermediary plateau could not be seen when the preincubationtime was 10 min and the activation was completed at 10 mM P_i.RuBisCO from Euglena also showed a quite similar activationkinetics. The activation was not due to the contaminating CO₂included in the stock P_i solution or in the activation buffercontaining the enzyme. The experiments with 2-carboxyarabinitol1,5-bisphosphate showed that the P_i stimulated activation wasdue to the promotion of binding of the activator CO₂ to theactivation sites. It was also found that P_i increased the affinityof RuBisCO for the activator CO₂ 5.4-fold accompanied by a decreaseof the half-saturating concentration of CO₂ to 1.6 µMat 20 mM MgCl₂. Physiological significance of the effects ofP_i on the activation of RuBisCO is discussed. ²Present address: Laboratory of Plant Molecular Physiology,Research Institute of Innovative Technology for the Earth (RITE),9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, Japan.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Inflammatory pseudotumor of the lung diagnosed as granulomatous lesion by preoperative brushing cytology. A case report

The clinical and cytologic features of a case of inflammatory pseudotumor of the lung are presented. Chest roentgenograms revealed a solitary circumscribed round mass in a nine-year-old boy. The mass was diagnosed as a granulomatous lesion by bronchoscopic brushing cytology. Although smears and cultures of sputum and brushing specimens were negative for tuberculosis, a tuberculin reaction was positive and antitubercular therapy was instituted. Since the mass had grown further after six months of therapy, an open lung biopsy was performed to resect the lesion and establish the diagnosis. Imprint smears of the cut surface of the lesion showed cytologic features similar to those of the brushings: short, spindle-shaped cells with a tendency to be arranged in stori-form patterns against a background of minimal necrotic debris. Histopathology established the final diagnosis of inflammatory pseudotumor, a rare granulomatous lesion radiologically resembling a true tumor. Since this lesion usually occurs in younger patients, inflammatory pseudotumor should be considered in pediatric cases with an intrapulmonary lesion that shows histiocytic spindle-shaped cells in stori-form patterns, but whose smears and cultures test negative for tuberculosis.     

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture‐based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture‐based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.