Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Superinfection with R Factors by Transduction in Escherichia coli and Salmonella typhimurium

Superinfection immunity is found in the conjugal transfer of R factors between two fi(+) R factors and between two fi(-) R factors (fi = fertility inhibition), as we reported previously. In contrast, no reduction in the frequencies of transduction of an fi(+) R factor 222 was caused by the presence of fi(+) R factors in the recipients in transduction systems with phage P1kc in Escherichia coli K-12 and with phage P22 in Salmonella typhimurium LT-2. The absence of superinfection immunity in transduction may be due to the difference in the route of entry of the R factor. The frequencies of transduction of an fi(+) R factor were reduced, although slightly, by the presence of fi(-) R factors in the recipients. This reduction is probably due to host-controlled restriction of the entering fi(+) R factor by the fi(-) R factors in the recipients, since transduction of an fi(+) R factor by the transducing phage propagated on the strain carrying both fi(+) and fi(-) R factors was not reduced by the presence of homologous fi(-) R factors in the recipients. The fi(+) R factor 222, when transduced to the recipient strains carrying other R factors, recombined genetically at high frequencies with these resident R factors, regardless of their fi type.     

Nitrotyrosine formation and its role in various pathological conditions

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. In polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes or endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t1/2 was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Facile Method for the Preparation of 7-Methyl-8-oxoguanosines as an Immunomodulator¶

Abstract

Reaction of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

     

Karyotypic evolution in the Galliformes: an examination of the process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny

To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst's pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1-9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented.     

A comparative karyological study of the blue-breasted quail (Coturnix chinensis, Phasianidae) and California quail (Callipepla californica, Odontophoridae)

We conducted comparative chromosome painting and chromosome mapping with chicken DNA probes against the blue-breasted quail (Coturnix chinensis, CCH) and California quail (Callipepla californica, CCA), which are classified into the Old World quail and the New World quail, respectively. Each chicken probe of chromosomes 1-9 and Z painted a pair of chromosomes in the blue-breasted quail. In California quail, chicken chromosome 2 probe painted chromosomes 3 and 6, and chicken chromosome 4 probe painted chromosomes 4 and a pair of microchromosomes. Comparison of the cytogenetic maps of the two quail species with those of chicken and Japanese quail revealed that there are several intrachromosomal rearrangements, pericentric and/or paracentric inversions, in chromosomes 1, 2 and 4 between chicken and the Old World quail. In addition, a pericentric inversion was found in chromosome 8 between chicken and the three quail species. Ordering of the Z-linked DNA clones revealed the presence of multiple rearrangements in the Z chromosomes of the three quail species. Comparing these results with the molecular phylogeny of Galliformes species, it was also cytogenetically supported that the New World quail is classified into a different clade from the lineage containing chicken and the Old World quail.     

Suppression of anoikis by v-Src but not by activated c-H-ras in human gallbladder epithelial cells

Detachment of anchorage-dependent normal epithelial cells from their substratum causes the type of apoptosis known as anoikis, whereas malignant cells can proliferate independently of anchorage. Because src and ras oncogenes are activated in many human cancers, we investigated their role and downstream signaling pathways in anoikis resistance, using HAG-1 human epithelial cells transfected with v-src or activated H-ras. Consequently, anchorage-dependent mock- or ras-transfected cells underwent anoikis. In contrast, anchorage-independent v-Src-transformed cells did not exhibit such apoptotic features. Focal adhesion kinase (FAK), a transducer of integrin, was only activated in v-Src-transformed cells. Herbimycin A, an Src kinase inhibitor, reduced tyrosyl phosphorylation of FAK and reversed resistance to anoikis. However, both protein kinase C (PKC) and phophatidylinositol-3 (PI-3) kinase inhibitors failed to induce anoikis. These data suggest that the ability of activated Src to prevent anoikis may be mediated by Src to a downstream signaling pathway involving FAK, but not Ras, PI-3 kinase, or PKC.     

Interchromosomal duplication of major histocompatibility complex class I regions in rainbow trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid ancestry

Salmonid fishes are among the few animal taxa with a probable recent tetraploid ancestor. The present study is the first to compare large (>100 kb) duplicated genomic sequence fragments in such species. Two contiguous stretches with major histocompatibility complex (MHC) class I genes were detected in a rainbow trout BAC library, mapped and sequenced. The MHC class I duplicated regions, mapped by fluorescence in situ hybridization (FISH), were shown to be located on different metaphase chromosomes, Chr 14 and 18. Gene organization in both duplications is similar to that in other fishes, in that the class I loci are tightly linked with the PSMB8, PSMB9, PSMB10 and ABCB3 genes. Whereas one region, Onmy-IA, has a classical MHC class I locus (UBA), Onmy-IB encodes only non-classical class Ib proteins. The nucleotide diversity between the Onmy-IA and Onmy-IB noncoding regions is about 14%. This suggests that the MHC class I duplication event has occurred about 60 mya close to the time of an hypothesized ancestral tetraploid event. The present article is the first convincing report on the co-existence of two closely related MHC class I core regions on two different chromosomes. The interchromosomal duplication and the homology levels are supportive of the tetraploid model.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB162342, AB162343 and from AY525774 to AY525776.     

The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.