In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma cells.     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Divergence between the Anoas of Sulawesi and the Asiatic Water buffaloes, inferred from their complete amino acid sequences of hemoglobin ß chains

Four complete amino acid sequences of hemoglobin β chains were determined for the swamp and the river types of the Asiatic water buffalo (Bubalus bubalis) and two species of the subgenus Anoa in Bubalus; B. (A.) depressicornis (H. Smith, 1827), the lowland anoa, and B. (A.) quarlesi (Ouwens, 1910), the mountain anoa. The two types of the bubalis were identical in the 145 amino acid residues of the β chains and, compared to this sequence, the two residues were substituted in the depressicornis (β49Thr → Ser and 134Ala → Thr) and the five were in the quarlesi (β53Val → Ile, 74Met → Ile, 111Val → Ile, 115Arg → His and 134Ala → Thr). While both Anoa species diverged from the bubalis by the β134Ala → Thr, they differed from each other by the five substitutions. The Anoa species are endemic to Sulawesi of Indonesia. Their speciation and the present coexistence were discussed with reference to probable immigrations of two ancestral Anoa species to Sulawesi at so long interval that had caused a reproductive isolation between the two wild animals. The earlier immigrants were postulated to be ancestral to the quarlesi and the later ones to the depressicornis.     

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek''s disease virus

The Marek's disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gplOO by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gplOO is not necessary for transport of gB to the cell surface.     

Regional changes in muscle mass and strength following 20 days of bed rest, and the effects on orthostatic tolerance capacity in young subjects

To this day, many studies have suggested that prolonged bed rest (BR) affects on muscle mass and strength not only in gravity muscles but also in ungravity muscles. However, it is still unclear whether the decrease in regional muscle strength after BR is due to the alterations in the corresponding muscle mass, or not. On the other hand, if BR decreases the mass of antigravity muscles (UGM) as well as muscle strength and then increases tissue compliance of the antigravity muscles, orthostatic tolerance capacity will be decreased by the reduction in cardiac output (CO) in spite of the increase in myocardial contractility because the more decrease in venous return due to the more increase in blood pooling within the compliant tissues of the lower body. However, this is also unclear. To make these questions clear, the present study investigated the regional muscle mass and strength and orthostatic tolerance capacity before and after 20 days of bed rest in young subjects.     

Mechanism of inhibitory action of capsaicin on particulate axoplasmic transport in sensory neurons in culture

The inhibitory effect of capsaicin on axoplasmic transport in cultured dorsal root ganglion cells was analyzed by video-enhanced contrast microscopy. Capsaicin inhibited particle transports in a dose-dependent manner, irrespective of the diameter of axons. The effect of capsaicin was reversible at low concentrations. Capsaicin affected both the anterograde and retrograde transport. Large organelles were more sensitive to capsaicin than small ones in the retrograde transport. An experiment using calcium-sensitive dye, Fura 2, indicated that capsaicin raised the intraneuronal free calcium concentration preceding the inhibition of the transport. Electron microscopy revealed that microtubules and neurofilaments are disorganized and disoriented by capsaicin. We reached a conclusion that capsaicin inhibits fast axoplasmic transport of both anterograde and retrograde directions in all types of somatosensory neurons in culture by disorganizing intraaxonal cytoskeletal structures, through the elevated intracellular Ca²⁺ concentration. © 1993 John Wiley & Sons, Inc.     

New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.