Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Vegetative diversification and radiation in subtribeDendrobiinae (Orchidaceae): Evidence from chloroplast DNA phylogeny and anatomical characters

The data derived from a chloroplast DNA restriction site analysis of subtribeDendrobiinae (Orchidaceae) indicate that extreme vegetative diversification is concentrated in two limited parts of this group. Overlaying the vegetative character states onto the chloroplast DNA cladogram suggests that several xeromorphic, vegetative characters evolved in the lines leading to the above-mentioned clades. Several anatomical characters are also associated with xeromorphy. These vegetative and anatomical characters facilitated the establishment of this group in various dry habitats. On the other hand, the modifications of size and number of parenchymatous cells substantially contributed to the vegetative diversification. This fact implies that a simple structural adjustment can result in a major modification of growth habits in theDendrobiinae.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Phencyclidine-induced expression of c-Fos-like immunoreactivity in mouse brain regions

For the purpose of studying a role of immediate early genes in psychotomimetic-induced behavioral excitation, we experimentally enhanced the locomotor activity of mice by acute administration of phencyclidine and examined the expression and localization of the c-Fos-like and c-Jun-like immunoreactivities in brain regions. A single injection of phencyclidine (5.0 mg/kg, i.p.) significantly increased not only the locomotor activity but also the expression of c-Fos-like immunoreactivity in several brain regions, particularly in the parietal cortex, hippocampal dentate gyrus, piriform cortex and hypothalamus. Interestingly, the c-Fos-like immunoreactivity in the parietal cortex continued to increase for 1 week after the phencyclidine injection. These results indicate that phencyclidine, even injected only once, can induce the persistent expression of c-Fos or c-Fos-related protein(s) in the mouse brain, and also suggest the possibility that such a c-Fos expression may underlie the behavioral and/or psychotomimetic effects of phencyclidine.     

Mechanism of Electron Flow through the QB Site in Photosystem II. 4. Reaction Mechanism of Plastoquinone Derivatives at the QB Site in Spinach Photosystem II Membrane Fragments

Interactions of externally added plastoquinone (PQ) derivatives(PQ₀-PQ₃) with the photosystem II (PSII) acceptor side wereinvestigated in PSII membrane fragments prepared from spinachby measuring the photoreduction rates of PQ derivatives at variousPQ concentrations, and the following results were obtained. From the kinetic analysis, all the PQ derivatives (PQ₀-PQ₃)except PQ₃ were shown to accept electrons at two sites (theQ_B site and the PQ site) as in the case of Synechococcus vulcanusPSII particles with benzoquinone derivatives [Satoh et al. (1995)Plant Cell Physiol. 36: 597]. Affinities of PQ derivatives at the Q_B site increased as thelength of the isoprene side chain got longer, while those atthe PQ site were not very much different for all the PQ derivativestested in this study. The inhibitory effect of DCMU was noncompetitive, and, therefore,the affinity of PQ₃ for the PQ site was determined while thatfor the Q_B site could not be estimated presumably due to itsfairly high affinity to the site. Based on the results obtained using PQ derivatives, the mechanismof interaction of an authentic PQ, PQ₉, at the Q_B site is discussed. (Received May 2, 1996; Accepted July 24, 1996)     

Assignment of the CD45-AP Gene to the Centromeric End of Mouse Chromosome 19 and Human Chromosome 11q13.1–q13.3

CD45-AP is a recently identified phosphorylated protein that specifically associates with the leukocyte-specific transmembrane glycoprotein CD45. The gene for CD45-AP,Ptprcap(protein tyrosine phosphatase, receptor type c polypeptide associated protein), was mapped in mouse by typing the progeny of two multilocus crosses using the mouse CD45-AP cDNA as a Southern hybridization probe. The CD45-AP gene mapped to the centromeric region of Chr 19 proximal to the genesFth, Cd5,andPcna-rs.The gene for the human CD45-AP homologue,PTPRCAP,was localized to chromosome band 11q13.1–q13.3 by fluorescencein situhybridization using human genomic CD45-AP DNA as a hybridization probe. The genetic mapping of thePtprcap/PTPRCAPgenes extends the previously defined synteny conservation of various genes that have been assigned to these regions of the mouse and the human chromosomes.     

Application of radiotelemetry to the survey of acorn dispersal byApodemus mice

We examined the applicability of radiotelemetry to studies of acorn dispersal byApodemus mice and compared its efficiency with the of this spool-and-line method. Installation of a transmitter (2.2 g) onto acorns did not interfere with the transporting and feeding behavior of the mice. We were able to detect all transmitter-installed acorns and follow the daily changes in the sites in which they were hoarded, while we missed 59% of the spool-tied acorns due to mice breaking the threads. Mice carried transmitter-installed acorns farther than spool-tied ones. The radiotelemetry method is superior to the spool-and-line method and useful for the study of hoarding behavior in rodents.