Karyology of the primitive slamanders,family hynobiidae

Summary Karyotypes have been studied in 3 species ofHynobius and in 1 species each of the remaining genera of Hynobiids (Ranodon, Batrachuperus, Salamandrella andOnychodactylus). All species have large diploid numbers, between 56 and 66, and asymmetrical and bimodal karyotypes. DNA contents (2C) were found to vary between 33 and 51 pg. Determination was not possible inOnychodactylus where higher values may be suspected. Some of the karyotypes investigated are similar to those of Cryptobranchids. Phylogenetic implications are discussed.Research supported by a grant from the Ministero della Pubblica Istruzione.     

Cytogenetic studies of Hynobiidae (Urodela). IX. Karyological characteristics ofHynobius abei Sato by means of R-and C-banding

Summary Chromosomal characteristics of the salamander speciesHynobius abei, from Ohimya (Kyoto) were revealed by the techniques of R-and C-banding. The karyotype ofH. abei was characterized by the shortness of an R-negative (C-positive) band in the terminal region of the long arm of chromosome 2 and a band encompassing the whole short arm of chromosome 10. These two bands inH. abei were the shortest among those of the variousHynobius species that have been examined. Otherwise no differences could be detected betweenH. abei and seven other pond-type species ofHynobius (2n=56) in terms of the banding patterns of 18 specifically identifiable pairs of their chromosomes.     

Karyotype of the Japanese salamander,Hynobius abei

Summary A primitive representative of the Caudata endemic to Japan,Hynobius abei Sato (Caudata: Hynobiidae) has 2n=56 chromosomes, with 9 large, 4 medium and 15 small-sized homologous pairs. The morphology of the large-sized chromosomes is similar to that of the knownHynobius species, but the presence of a pair of acrocentrics in the medium-sized group and 5 pairs of biarmed chromosomes in the small-sized group characterized the karyotype ofH. abei.We thank A. Itoi, S. Segawa, K. Ban, T. Hikida and O. Murakami for their assistance in collecting specimens.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997     

A role for the hinge/ear domain of the β chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the β2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated β2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the β chains indeed provide an important interaction between the AP complexes and clathrin. Received 7 April 1997; received after revision 22 May 1997; accepted 28 May 1997     

Research Articles¶Naturally occurring 2′-O-methylpurine nucleosides with hypotensive properties

2′-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor, 2′-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine. Received 2 October 1997; accepted 28 October 1997     

Biotransformation of the flavonoid tiliroside to 7-methylether tiliroside: bioactivity of this metabolite and of its acetylated derivative

Incubation of kaempferol-3-O-β-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) with Aspergillus nidulans gives the 7-methyl ether of tiliroside (2) which is a new compound. Its structure is determined by spectroscopic methods. Cytotoxic studies of 2 and of its acetylated derivative 2a were carried out in vitro against fourteen human leukemic cell lines. Results clearly show that compound 2 is ineffective against all leukemic cell lines tested. On the contrary, compound 2a exhibited cytotoxic activity against four of the cell lines (HL60, DAUDI, HUT78 and MOLT3) and additionally, a dose- and time-dependent effect on DNA synthesis. Received 18 February 1997; received after revision 8 April 1997; accepted 6 May 1997     

Interaction of Co, Mn, Mg and Al with d(GCCCATGGC) and d(CCGGGCCCGG): a spectroscopic study

Spectroscopic study of the interactions of metal ions, Co, Mn, Mg and Al with d(GCCCATGGGC) and d(CCGGGCCCGG) revealed the following. Metal ions Mn, Al and Mg at the lowest concentrations enhanced the t _m of oligomers, whereas Mn and Mg at higher concentrations decreased the t _m . Co enhanced the t _m of oligomers at higher concentrations. The studies have also indicated that Mn at lower concentrations displaced EtBr fluorescence, Mg and Co at moderate concentrations and Al only at higher concentrations. Addition of Co, Mn, Mg and Al altered the bands of the circulars dichroism (CD) spectra of the oligomers in a concentration-dependent manner. The CD spectra of d(GCCCATGGGC) and d(CCGGGCCCGG) indicated B and Z forms of DNA, respectively, in contrast to the A form observed in the crystal structures. Mg and Co at different ionic strength induced Z–B transition in d(CCGGGCCCGG), while Al at higher concentrations induced a Z–A transition. Mn did not induce any transition. This is the first report to show that Al causes structural transitions in sequence-specific oligomers and has strong binding ability with GC-rich euchromatin oligomers. Received 22 December 1997; received after revision 16 March 1998; accepted 16 March 1998     

The karyotype,C-bands and AgNO3-bands of a lungless salamander from Korea:Onychodactylus fischeri (Boulenger) (Amphibia,Urodela)

The karyotype of a lungless salamander,Onychodactylus fischeri, from Korea was analyzed and compared with that of the Japanese congeneric species,O. japonicus. In both species the diploid karyotype consists of78 chromosomes, including 6 pairs of large chromosomes, 6 pairs of medium-sized ones, and the remaining 27 pairs of microchromosomes. The chromosome number ofO. fischeri, 2n=78, is, like that ofO. japonicus, the largest so far reported in the order Urodela. C-banding showed that constitutive heterochromatin inO. fischeri was mainly in the centromeric regions and near the secondary constrictions of the large chromosomes. AgNO₃-bands were located in the secondary constrictions associated with C-band heterochromatin.     

Chromosomal evolution and zoogeographic origin of southeast Asian shrews (genusCrocidura)

To the origins and evolution of Indomalayan shrews, we investigated the chromosomal variations of 14 species ofCrocidura from SE Asia. Intraspecific polymorphism was mainly due to variation in the number of short chromosomal arms butC. lepidura andC. hutanis showed a polymorphism due to a centric fusion. The undifferentially stained karyotypes were similar in 9 species, all possessing 2n=38 and FN=54–56 (68);C. fuliginosa had 2n=40 and FN=54–58. These karyotypes are close to the presumed ancestral state for the genusCrocidura. Four species from Sulawesi had a reduced diploid number (2n=30–34), a trend not observed among other SE Asian species but present in few Palaearctic taxa. Compared to the apparent stasis of karyotypic evolution observed among other SE Asian species, the high degree of interspecific differences reported among Sulawesian shrews is unusual and needs further investigation.Stasis and reduction in diploid number found in both Indomalayan and Palaearctic species suggest that these two groups share a common ancestry. This is in sharp contrast to most Afrotropical species which evolved towards higher diploid and fundamental numbers. The zoogeographical implications of these results are discussed.     

Drosophila unconventional myosin VI is involved in intra- and intercellular transport during oogenesis

During mid-oogenesis of Drosophila, cyto plasmic particles are transported within the nurse cells and through ring canals (cytoplasmic bridges) into the oocyte by means of a microfilament-dependent mecha nism. Video-intensified fluorescence timelapse mi croscopy, in combination with microinjections of antibodies directed against Drosophila 95F myosin, have revealed that this unconventional myosin of class VI is involved in the transport processes. The results indicate that certain cytoplasmic particles in the nurse cells move along microfilaments due to their direct association with myosin VI motors. Additional myosin- VI molecules located at the rim of the ring canals seem to be involved in particle transport into the oocyte. Microinjected mitochondria-specific dyes have revealed that some of these particles are mitochondria. Received 3 April 1997; received after revision 5 May 1997; accepted 27 May 1997     

Biochemical genetic differentiation among seed chalcid species of genusMegastigmus (Hymenoptera: Torymidae)

The degree of genetic variability and the taxonomic status of adults of 14 seed chalcid species of the genusMegastigmus was analyzed using electrophoresis on horizontal starch gel. Variability of chalcid populations with host tree was observed in 21 host species. A total of 13 enzyme loci were considered. Seven of the loci were found to be polymorphic. The electrophoretic data strongly supported the adaptation of each chalcid species to a limited number of congeneric hosts, and confirmed the morphologically-based taxonomy of the genus. The resulting dendrogram separated the chalcid species into three distinct groups, infesting 1) Pinaceae spp., 2) Cupressaceae spp., and 3) Angiosperm spp., respectively. The highest level of overall genetic similarity was observed among the chalcid populations infesting conifers of the generaPseudotsuga andAbies. The genetic identity values observed among populations infesting 5 differentAbies species tended to reflect the occurrence of conspecific populations rather than that of distinct chalcid species. Genetic identity was similarly important among the chalcid species infesting seeds of Cupressaceae. By contrast, a large genetic distance was observed between two seed chalcids attacking a same host,Rosa montana.     

A new chromosome number forBombina (Anura,Discoglossidae)

Summary The karyotype of a primitive discoglossid anuran,Bombina maxima, native to southwestern China, has 2n=28 chromosomes with 6 large and 8 small bi-armed homologous pairs. This is a higher chromosome number than described for otherBombina species, all of which have 2n=24.     

Bioassay for hamster macrophage chemotaxis: application to study particle-lung interactions

Attraction of lung macrophages to particle deposition sites has been demonstrated in different animal species. We reported a threefold increase of the number of macrophages to occur within 40 min after polystyrene particle deposition in hamster airways [Geiser et al. (1994) Am. J. Respir. Cell Mol. Biol. 160: 594–603]. Complement-derived chemotactic activity is one of the mechanisms postulated for macrophage recruitment. It was the aim of this study to test whether complement-derived chemotactic activity is involved in the rapid recruitment of macrophages to the site of deposited polystyrene particles in hamster airways. We first developed an in vitro cell migration assay for hamster macrophages to assess complement-derived chemotaxis. Second, the bronchoalveolar lavage fluids (BALF) of four hamsters that had inhaled aerosols of polystyrene microspheres were tested for chemotactic activity by this bioassay and compared with BALF of four sham-exposed hamsters. Chemotactic response of macrophages was found toward complement-activated hamster serum, whereas macrophage migration was not increased toward BALF of particle and sham-exposed hamsters. In contrast, macrophage migration to BALF of both groups was reduced by 1.6-fold. Thus, the stimulus for macrophage recruitment to the site of deposited polystyrene particles in hamster airways could not be demonstrated using this bioassay. Received 10 September 1997; received after revision 24 November 1997; accepted 10 December 1997     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

Karyology of the primitive salamanders, family Hynobiidae

Karyotypes have been studied in 3 species of Hynobius and in 1 species each of the remaining genera of Hynobiids (Ranodon, Batrachuperus, Salamandrella and Onychodactylus). All species have large diploid numbers, between 56 and 66, and asymmetrical and bimodal karyotypes. DNA contents (2C) were found to vary between 33 and 51 pg. Determination was not possible in Onychodactylus where higher values may be suspected. Some of the karyotypes investigated are similar to those of Cryptobranchids. Phylogenetic implications are discussed.     

Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish. Received 22 December 1997; received after revision 5 March 1998; accepted 5 March 1998