Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.     

Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5-10 microM) resulted in a dose-dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on attachment to fibronectin. Phorbol 12-myristate 13-acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.     

Chromosome studies in the mammalian subfamily Antilopinae

The karyology was studied in nine species of Antilopinae and evaluated with regard to cytotaxonomic relations within the subfamily. Karyotypes of three of these species were previously undescribed. Chromosomes were examined by conventional staining methods, G-, C-, and T-banding techniques, and by autoradiography. Evolutionary differentiation of karyotypes in this group is characterized by extensive Robertsonian fusions and a particular translocation between the X chromosome and an autosome. With comparison of Giemsa-banding patterns a taxonomy has been constructed which differs most markedly from the classical taxonomy in two aspects: the blackbuck, Antilope cervicapra, shows a strong karyotypic affinity to gazelles of the subgenus Nanger; Thomson's gazelle, Gazella thomsoni, lacks the numerous Robertsonian fusions and the X-autosome translocation common to other members of Gazella studied to date. Cases of intraspecific polymorphism of chromosome morphology and number are presented.     

The phylogeny of the ant tribe Formicini (Hymenoptera: Formicidae) with the description of a new genus

Abstract. The holarctic ant tribe Formicini is revised, the new genus Bajcaridris described, and possible phylogenetic relationships are discussed. The subgenus Iberoformica is synonymized with Formica. A synopsis, diagnosis and keys to the genera are provided.     

Synthesis of 2-Alkynylcordycepins and Evaluation of Their Vasodilating Activity

Abstract

Based on the recently developed lithiation-mediated stannyl migration of 6-chloropurine derivatives, 2-iodocordycepin was prepared from cordycepin. The reaction of this compound with terminal alkynes was carried out to synthesize a series of 2-alkynyl derivatives. The vasodilating effect of these compounds was evaluated.     

Developmental change and sexual difference in synaptic modulation produced by oxytocin in rat substantia gelatinosa neurons

We have previously reported that oxytocin produces an inward current at a holding potential of ?70 mV without a change in glutamatergic excitatory transmission in adult male rat spinal lamina II (substantia gelatinosa; SG) neurons that play a pivotal role in regulating nociceptive transmission. Oxytocin also enhanced GABAergic and glycinergic spontaneous inhibitory transmissions in a manner sensitive to a voltage-gated Na⁺-channel blocker tetrodotoxin. These actions were mediated by oxytocin-receptor activation. Such a result was different from that obtained by other investigators in young male rat superficial dorsal horn neurons in which an oxytocin-receptor agonist enhanced glutamatergic and GABAergic but not glycinergic spontaneous transmissions. In order to know a developmental change and also sexual difference in the actions of oxytocin, we examined its effect on spontaneous synaptic transmission in adult female and young male rat SG neurons by using the whole-cell patch-clamp technique in spinal cord slices. In adult female rats, oxytocin produced an inward current at ?70 mV without a change in excitatory transmission. GABAergic and glycinergic transmissions were enhanced by oxytocin, the duration of which enhancement was much shorter than in adult male rats. In young (11–21 postnatal days) male rats, oxytocin produced not only an inward but also outward current at ?70 mV, and presynaptically inhibited or facilitated excitatory transmission, depending on the neurons tested; both GABAergic and glycinergic transmissions were enhanced by oxytocin. The inhibitory transmission enhancements in adult female and young male rats were sensitive to tetrodotoxin. Although the data may not be enough to be estimated, it is suggested that synaptic modulation by oxytocin in SG neurons, i.e., cellular mechanism for its antinociceptive action, exhibits a developmental change and sexual difference.     

Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC-EPR and HPLC-EPR-MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography-electron paramagnetic resonance spectrometry (HPLC-EPR) and high performance liquid chromatography-electron paramagnetic resonance-mass spectrometry (HPLC-EPR-MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC-EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC-EPR-MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.