Regulation of ethylene biosynthesis after short-term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short-term heal treatment and the regulation of heat-altered ethylene production. Subapical stem segments of 7-day-old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) oxidation, and ACC and l-(malonylamino)cyclopropane-l-carboxylic acid (MACC) content after a 2-min 40°C heat pulse. The short-term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30-min time period. Conjugation to MACC did not appear to play an integral role in heat-regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.     

Anticarcinogenesis in fish.

Rainbow trout respond to a range of natural and synthetic dietary tumor modulators. Observed modulations of final tumor incidence include inhibition, promotion and cocarcinogenesis, depending on modulator, carcinogen, target organ, and relative order of carcinogen and modulator exposure. Despite several obvious limitations (e.g. lung, colon, mammary gland, bladder are not available as target organs), the trout model possesses several important features that have made it valuable for tumor modulation studies. (1) The comparative advantage. Since rodents are not perfect human surrogates, studies with alternative vertebrates such as trout have provided important comparative mechanism information for confident extrapolation of animal studies to humans. For example, beta-naphthoflavone appears to inhibit aflatoxin B1 hepatocarcinogenicity by species-independent mechanisms that readily extrapolate to humans. By contrast other modulators, including butylated hydroxyanisole, inhibit aflatoxin B1 hepatocarcinogenesis in rats by an enabling mechanism shown to be absent in trout, namely the induction of an aflatoxin B1-specific glutathione S-transferase isozyme. Interestingly, evidence is presently lacking that this determinant mechanism would be operative in humans. (2) The sensitivity advantage. Trout sensitivity and small body size at exposure have permitted tumor studies with carcinogens and HPLC-purified anticarcinogen intermediates too scarce to study in rodents. (3) The low cost advantage. The very low cost of trout tumor studies has enabled statistically challenging issues in molecular dosimetry, dose-response, and risk-benefit analysis to be addressed using as many as 9600 animals per tumor study at modest budget. In particular, these designs provide modulator-mediated alterations in precisely determined carcinogen TD50 values, rather than changes in simple tumor incidence, to quantify more rigorously modulator potencies for tumor inhibition or promotion as a function of dietary concentrations.     

Differential activation within costal diaphragm during rapid-eye-movement sleep in cats

Simultaneous recordings of the diaphragmatic electromyogram (EMG) were made from two separate regions of the costal diaphragm in six normal cats. The diaphragmatic activities were always synchronous and the amplitudes and rates of rise were similar during slow-wave sleep. In contrast, during natural rapid-eye-movement (REM) sleep, different activity was often present in the two leads. These differences were in the time of onset and offset, as well as in the amplitude and spike patterns, and occurred in approximately 5-20% of the diaphragmatic bursts averaged over the entire REM sleep period. With respect to eye movement density, the rate of differential activation was higher during periods of high density (26%) than in the absence of eye movements (1%) in the four animals for which these data were available. Differential activation of portions of the costal diaphragm is apparently a normal event of REM sleep. This could result from descending state-specific phasic neuronal activity that bypasses the medullary respiratory generator. Differential activation of portions of the diaphragm could contribute to disordered ventilation during REM sleep.     

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.     

IFN-gamma and IL-2 are protective in the skin but pathologic in the corneas of HSV-1-infected mice.

HSV type 1 (HSV-1) infection of the mouse cornea results in a tissue-destructive inflammatory reaction in the cornea, but little or no disease in the skin surrounding the eye. Depleting T lymphocytes from mice before HSV-1 corneal infection prevents the corneal inflammation but severely exacerbates the periocular skin lesions. Studies described in this communication investigated the role of T cell cytokines in the corneal and periocular skin disease induced by HSV-1 corneal infection. Mice received weekly i.p. injections of rat mAb specific for IL-2, IL-4, or IFN-gamma beginning 1 day before (day -1) or 6 days after (day +6) corneal infection with the RE strain of HSV-1. The severity of corneal inflammation and the area of periocular skin involvement were measured. Treatment with anti-IFN-gamma or anti-IL-2 significantly reduced the incidence and severity of corneal inflammation. Treatment was equally effective when initiated on day -1 (before T cell activation) or day +6 (after T cell activation but before the initiation of corneal inflammation). Treatment with anti-IL-4 had no effect. The histologic features of corneal inflammation in mock-treated mice included neovascularization, corneal edema, and cellular infiltration. Corneas of anti-IL-2-treated mice that developed inflammation had similar but less severe histologic features. Corneas of anti-IFN-gamma-treated mice that developed inflammation had neovascularization and edema but minimal cellular infiltration. Treatment with anti-IFN-gamma or anti-IL-2 significantly exacerbated periocular skin lesions when initiated at day -1, but not when initiated at day +6. Anti-IL-4 treatment had no effect on skin lesions. Treatment with either anti-IFN-gamma or anti-IL-2, when initiated at day -1, significantly inhibited the delayed-type hypersensitivity response to HSV Ag, but when treatment was begun at day +6 only anti-IFN-gamma significantly inhibited the delayed-type hypersensitivity response. Our findings suggest that IFN-gamma and IL-2 are important elements in both an immunopathologic T-lymphocyte response to HSV-1 Ag in the cornea and a protective T lymphocyte response in the skin.     

Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase

In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G₁ and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.     

Histone gene switch in the sea urchin embryo. Identification of late embryonic histone messenger ribonucleic acids and the control of their synthesis.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.     

A method for constraining the age of origination of derived characters

Fossils are the physical records of the history of morphological character evolution on Earth and can provide valuable information concerning the sequence and timing of origination of derived characters. Knowledge of the timing of origination of synapomorphies makes it possible to estimate when unobserved character changes occurred in the geological past. Here we present a method for estimating the temporal interval during which synapomorphies evolved. The method requires either direct inclusion of fossil taxa (with or without extant taxa) in cladistic analyses based on morphological or combined data, or indirectly using the “molecular scaffold approach.” Second, characters of interest are mapped on a most parsimonious tree and “minimum age node mapping” is used to place minimum ages on the nodes of the tree. Finally, characters of interest are evaluated for younger and/or older temporal constraints on the time of their origination; application of the older bound assumes ancestry of fossil terminals included in the tree. A key is provided herein describing the method. Among other applications, this approach has the potential to provide a powerful test of purported evolutionary cause–effect relationships. For example, the method has the ability to discover that derived characters of suggested adaptational significance may considerably pre‐date the cause(s) that are hypothesized to have favored their establishment. © The Willi Hennig Society 2007.     

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse × hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19—a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution ofancestral HSA19 segments in eight mammalian orders involving about 50 species.