Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket,Acheta domesticus (Orthoptera:Gryllidae)

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.     

Photosynthetic activity of developing leaves of Zea mays is less affected by heat stress than that of developed leaves

Various physiological and biochemical characters of a leaf change with stages of its ontogeny. It is likely that the photosynthetic functions of leaves of different ontogeny have different levels of heat tolerance. This study was initiated to analyze the photosynthetic heat tolerance of fully-developed, nearly-developed (more than 2/3 expanded) and developing (10–12 cm visible) leaves of two maize genotypes, F223 and F250. The results indicate that the photosynthetic CO₂ assimilation rate (P_n) of developing leaves was less affected by heat stress (42°C in the dark for 90 min) than that of developed leaves. The impaired P_n recovered within 24 h in the developing leaves, while the P_n of developed and nearly-developed leaves did not reach the non-stress level, even after 72 h. The P_n of the developed leaves of genotype F250 was less affected by heat stress than that of genotype F223. After heat stress, the slightly affected P_n of the developing leaf was associated with the almost unchanged photochemical efficiency of photosystem II (F_v/F_m) and the quantum yield of photosystem II electron transport. The chlorophylls a and b were degraded by heat stress; the degradation was pronounced in the developed leaves. As a result of heat stress, the antheraxanthin and zeaxanthin of the xanthophyll cycle accumulated in both the nearly-developed and developed leaves but not in the developing leaves. Injury to the plasma membrane due to heat stress was much less severe in developing leaves than that in the developed leaves. From the physiological characters which we determined it would appear that the P_n functions of the developing leaves are more resistant to heat stress than those of nearly-developed and developed leaves.     

Infrared Thermography and Ultrasonography to Indirectly Monitor the Influence of Liner Type and Overmilking on Teat Tissue Recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30–41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

Structurally distinct recognition motifs in lymphotoxin-beta receptor and CD40 for tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.     

Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.     

Intragastric ethanol infusion model for cellular and molecular studies of alcoholic liver disease

The intragastric alcohol infusion rat model (IAIRM) of alcoholic liver disease (ALD) has been utilized in various laboratories to study various aspects of ALD pathogenesis including oxidative stress, cytokine upregulation, hypoxic damage, apoptosis, ubiquitin-proteasome pathway and CYP2E1 induction. The basic value of the model is that it produces pathologic changes which resemble ALD including microvesicular and macrovesicular fat, megamitochondria, apoptosis, central lobular and pericellular fibrosis, portal fibrosis, bridging fibrosis, central necrosis, and mixed inflammatory infiltrate including PMNs and lymphocytes. The model is valuable because the diet and ethanol intake are totally under the control of the investigator. A steady state can be maintained with high or low blood alcohol levels for long periods. The cycling of the blood alcohol levels, when a constant infusion rate of alcohol is maintained, simulates binge drinking. Using this model the importance of dietary fat, especially the degree of saturation of the fatty acids on the induction of liver pathology, has been documented. The role of endotoxin, the Kupffer cell, TNFalpha, and NADPH oxidase have been demonstrated. The importance of 2E1 in oxidative stress induction has been shown using inhibitors of the isozyme. The importance of dietary iron in the pathogenesis of cirrhosis has been documented. Acetaldehyde has been shown to play a role in preventing liver pathology by preventing NFkappaB activation. Using the model, to maintain high blood alcohol levels is found to be necessary to demonstrate proteasomal peptidase inhibition. Ubiquitin synthesis is also inhibited at high blood alcohol levels in the IAIRM model. Oxidized proteins accumulate in the liver at high blood alcohol levels. Neoantigens derived from protein adducts formed with products of oxidation induce autoimmune mechanisms of liver injury. Thus, in many ways the model has revolutionized our understanding of the pathogenesis of ALD.     

Failure of neutralizing gp120 monoclonal antibodies to prevent HIV infection of choriocarcinoma-derived trophoblasts

Although placental trophoblasts, the only fetal cells in direct contact with infectious maternal blood, can be infected with HIV, the precise cause for the low transmission rate of virus across the placental barrier is unknown. One of the most common conjectures is that maternal anti-HIV antibodies (Abs) contribute to the protection of the fetus. This hypothesis has been tested in vitro by infecting the CD4-negative placental trophoblast line, BeWo, with HIV-1_IIIB in the presence of serial dilutions of neutralizing monoclonal Abs against the V3 loop (No. 694) or CD4-binding conformational domain (No. 588). The results, based on measurement of p24 production from virus-exposed cells, reveal that the titers of Abs, adequate in preventing the infection of control MT-4 T lymphocytes, were less effective in protecting trophoblasts. Furthermore, PCR analysis of HIV DNA formed after a single round of infection has shown no significant decrease in the number of viral copies in Ab-protected BeWo cells. An anti-HIV serum from a pregnant woman did also have no effect. Although our in vitro observations do not necessarily apply to the in vivo situation, the results suggest that the humoral immune response sustained by neutralizing Abs may be able to protect T lymphocytes, but not placental trophoblasts. The findings are consistent with recent clinical studies demonstrating a lack of correlation between the presence of neutralizing anti-HIV Abs in pregnant women and HIV transmission in utero.     

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.     

Not going with the flow: a comprehensive time‐calibrated phylogeny of dragonflies (Anisoptera: Odonata: Insecta) provides evidence for the role of lentic habitats on diversification

Ecological diversification of aquatic insects has long been suspected to have been driven by differences in freshwater habitats, which can be classified into flowing (lotic) waters and standing (lentic) waters. The contrasting characteristics of lotic and lentic freshwater systems imply different ecological constraints on their inhabitants. The ephemeral and discontinuous character of most lentic water bodies may encourage dispersal by lentic species in turn reducing geographical isolation among populations. Hence, speciation probability would be lower in lentic species. Here, we assess the impact of habitat use on diversification patterns in dragonflies (Anisoptera: Odonata). Based on the eight nuclear and mitochondrial genes, we inferred species diversification with a model‐based evolutionary framework, to account for rate variation through time and among lineages and to estimate the impact of larval habitat on the potentially nonrandom diversification among anisopteran groups. Ancestral state reconstruction revealed lotic fresh water systems as their original primary habitat, while lentic waters have been colonized independently in Aeshnidae, Corduliidae and Libellulidae. Furthermore, our results indicate a positive correlation of speciation and lentic habitat colonization by dragonflies: speciation rates increased in lentic Aeshnidae and Libellulidae, whereas they remain mostly uniform among lotic groups. This contradicts the hypothesis of inherently lower speciation in lentic groups and suggests species with larger ranges are more likely to diversify, perhaps due to higher probability of larger areas being dissected by geographical barriers. Furthermore, larger range sizes may comprise more habitat types, which could also promote speciation by providing additional niches, allowing the coexistence of emerging species.     

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.