Mutation-biased adaptation in a protein NK model

Evolutionary trends responsible for systematic differences in genome and proteome composition have been attributed to GC:AT mutation bias in the context of neutral evolution or to selection acting on genome composition. A possibility that has been ignored, presumably because it is part of neither the Modern Synthesis nor the Neutral Theory, is that mutation may impose a directional bias on adaptation. This possibility is explored here with simulations of the effect of a GC:AT bias on amino acid composition during adaptive walks on an abstract protein fitness landscape called an "NK" model. The results indicate that adaptation does not preclude mutation-biased evolution. In the complete absence of neutral evolution, a modest GC:AT bias of realistic magnitude can displace the trajectory of adaptation in a mutationally favored direction, to such a degree that amino acid composition is biased substantially and persistently. Thus, mutational explanations for evolved patterns need not presuppose neutral evolution.     

In vitro and in vivo kinetic handling of nitrite in blood: effects of varying hemoglobin oxygen saturation

Growing evidence suggests that nitrite, acting via reduction to nitric oxide by deoxyhemoglobin, may play an important role in local control of blood flow during hypoxia. To investigate the effect of hypoxia (65 Torr arterial Po(2)) on the kinetic properties of nitrite, a bolus injection of sodium nitrite (10 mg/kg iv) was given to normoxic or hypoxic newborn lambs, and the time course of plasma nitrite and methemoglobin (MetHb) concentrations was measured. The in vivo kinetics of nitrite disappearance from plasma were biphasic and were not affected by hypoxia. Changes in MetHb, a product of the nitrite-hemoglobin reaction, also did not differ with the level of oxygenation. Hypoxia potentiated the hypotensive effects of nitrite on pulmonary and systemic arterial pressures. The disappearance of nitrite from plasma was equivalent to the increase in MetHb on a molar basis. In contrast, nitrite metabolism in sheep blood in vitro resulted in more than one MetHb per nitrite equivalent under mid- and high-oxygenation conditions: oxyhemoglobin (HbO(2)) saturation = 50.3 +/- 1.7% and 97.0 +/- 1.3%, respectively. Under the low-oxygenation condition (HbO(2) saturation = 5.2 +/- 0.9%), significantly less than 1 mol of MetHb was produced per nitrite equivalent, indicating that a significant portion of nitrite is metabolized through pathways that do not produce MetHb. These data support the idea that the vasodilating effects of nitrite are potentiated under hypoxic conditions due to the reduction of nitrite to nitric oxide by deoxyhemoglobin.     

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells.

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA's of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.     

NF-kappa B is required within the innate immune system to inhibit microflora-induced colitis and expression of IL-12 p40

We have previously presented evidence demonstrating that mice deficient in NF-kappaB subunits are susceptible to colitis induced by the pathogenic enterohepatic Helicobacter species, H. hepaticus. However, it has not been determined whether NF-kappaB is required within inhibitory lymphocyte populations, within cells of the innate immune system, or both, to suppress inflammation. To examine these issues, we have performed a series of adoptive transfer experiments using recombination-activating gene (Rag)-2(-/-) or p50(-/-)p65(+/-)Rag-2(-/-) mice as hosts for wild-type (WT) and p50(-/-)p65(+/-) lymphocyte populations. We have shown that although the ability of H. hepaticus to induce colitis in Rag-2(-/-) mice is inhibited by the presence of either WT or p50(-/-)p65(+/-) splenocytes, these splenocyte populations are unable to suppress H. hepaticus-induced colitis in p50(-/-)p65(+/-)Rag-2(-/-) mice. Colitis in these animals is characterized by increased expression of inflammatory cytokines including IL-12 p40, and depletion of IL-12 p40 from p50(-/-)p65(+/-) mice ameliorates H. hepaticus-induced disease. Consistent with a primary defect in the regulation of IL-12 expression, H. hepaticus induced markedly higher levels of IL-12 p40 in p50(-/-)p65(+/-) macrophages than in WT macrophages. These results suggest that inhibition of H. hepaticus-induced IL-12 p40 expression by NF-kappaB subunits is critical to preventing colonic inflammation in response to inflammatory microflora.     

A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain

Optical imaging has great potential for studying molecular recognitions both in vivo and in vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2, called DLIA-IL11Ralpha. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11Ralpha), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11Ralpha after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in vivo.     

Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth

For over 7 centuries, production of rice (Oryza sativa L.) in Egypt has benefited from rotation with Egyptian berseem clover (Trifolium alexandrinum). The nitrogen supplied by this rotation replaces 25- 33% of the recommended rate of fertilizer-N application for rice production. This benefit to the rice cannot be explained solely by an increased availability of fixed N through mineralization of N- rich clover crop residues. Since rice normally supports a diverse microbial community of internal root colonists, we have examined the possibility that the clover symbiont, Rhizobium leguminosarum bv. trifolii colonizes rice roots endophytically in fields where these crops are rotated, and if so, whether this novel plant-microbe association benefits rice growth. MPN plant infection studies were performed on macerates of surface-sterilized rice roots inoculated on T. alexandrinum as the legume trap host. The results indicated that the root interior of rice grown in fields rotated with clover in the Nile Delta contained 10⁶ clover-nodulating rhizobial endophytes g fresh weight of root. Plant tests plus microscopical, cultural, biochemical, and molecular structure studies indicated that the numerically dominant isolates of clover-nodulating rice endophytes represent 3 – 4 authentic strains of R. leguminosarum bv. trifolii that were Nod Fix on berseem clover. Pure cultures of selected strains were able to colonize the interior of rice roots grown under gnotobiotic conditions. These rice endophytes were reisolated from surface-sterilized roots and shown by molecular methods to be the same as the original inoculant strains, thus verifying Koch's postulates. Two endophytic strains of R. leguminosarum bv. trifolii significantly increased shoot and root growth of rice in growth chamber experiments, and grain yield plus agronomic fertilizer N-use efficiency of Giza-175 hybrid rice in a field inoculation experiment conducted in the Nile Delta. Thus, fields where rice has been grown in rotation with clover since antiquity contain Fix strains of R. leguminosarum bv. trifolii that naturally colonize the rice root interior, and these true rhizobial endophytes have the potential to promote rice growth and productivity under laboratory and field conditions.     

Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen

The extension of nitrogen-fixing symbioses to important crop plants such as the cereals has been a long-standing goal in the field of biological nitrogen fixation. One of the approaches that has been used to try to achieve this goal involves the isolation and characterization of stable endophytic bacteria from a variety of wild and cultivated rice species that either have a natural ability to fix nitrogen or can be engineered to do so. Here we present the results of our first screening effort for rice endophytes and their characterization using acetylene reduction assays (ARA), genomic fingerprinting with primers corresponding to naturally occurring repetitive DNA elements (rep-PCR), partial 16S rDNA sequence analysis and PCR mediated detection of nitrogen fixation (nif) genes with universal nif primers developed in our laboratory. We also describe our efforts to inoculate rice plants with the isolates obtained from the screening, in order to examine their invasiveness and persistence (stable endophytic maintenance). Lastly, we review our attempts to tag selected isolates with reporter genes/proteins, such as beta-glucuronidase (gus) or green fluorescent protein (gfp), in order to be able to track putative endophytes during colonization of rice tissues.